Cell-free production of ribonucleic acid

ABSTRACT

Provided herein, in some aspects, are methods and compositions for cell-free production of ribonucleic acid.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/319,220 filed Apr. 6, 2016 and U.S.provisional application No. 62/452,550 filed Jan. 31, 2017, each ofwhich is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Ribonucleic acid (RNA) is ubiquitous to life. RNA acts as the keymessenger of information in cells, carrying the instructions from DNAfor the regulation and synthesis of proteins. RNA is of interest inbiotechnology as synthetically modulating mRNA levels in cells(positively through the introduction of mRNA or negatively through theintroduction of siRNA or dsRNA) has applications in fields such asagricultural crop protection, anti-cancer therapeutics, and vaccines.RNA interference (RNAi), for example, refers to a cellular mechanismthat uses the DNA sequence of a gene to turn the gene “off”—a processreferred to as “silencing.” In a wide variety of organisms, includinganimals, plants, and fungi, RNAi is triggered by double-stranded RNA(dsRNA). Functional single-stranded (e.g. mRNA) and double-stranded RNAmolecules have been produced in living cells and in vitro usingpurified, recombinant enzymes and purified nucleotide triphosphates(see, e.g., European Patent No. 1631675 and U.S. Patent ApplicationPublication No. 2014/0271559 A1, each of which is incorporated herein byreference). Nonetheless, the production of RNA at scales enablingwidespread commercial application is currently cost-prohibitive.

SUMMARY OF THE INVENTION

Provided herein are methods, compositions, cells, constructs, andsystems for the production (biosynthesis) of RNA. Generally, polymericRNA from a biomass material is enzymatically depolymerized into itsconstituent monomers, these monomers are then phosphorylated to theircognate triphosphorylated variants (via a series of kinases) which aresubsequently polymerized into a polymeric RNA using a correspondingnuclei acid (e.g., DNA) template.

In some embodiments, the methods, compositions, cells, constructs, andsystems of the present disclosure are used for the production of RNAunder cell-free conditions, for example, using at least one cell lysate,a combination of purified proteins, or a combination of cell lysate(s)and purified protein(s). The present disclosure is based, in someembodiments, on the conversion of RNA from biomass (e.g., endogenouscellular RNA) to desired synthetic RNA (e.g., synthetic single-strandedor double-stranded RNA) using a cell lysate. First, RNA from biomass(e.g., endogenous RNA), such as messenger RNA (mRNA), transfer RNA(tRNA), and/or ribosomal RNA (rRNA) (e.g., present in a cell lysate) isdepolymerized into its monomeric form, 5′-nucleoside monophosphates(NMPs) by one or more nucleases (FIG. 1, reaction 1). Next, thesenucleases, as well as native nucleases and phosphatases, are inactivatedor partially inactivated (e.g., via heat inactivation), and the NMPs arephosphorylated to ribonucleotide triphosphates (NTPs) by a series ofthermostable kinase activities (FIG. 1, reaction 2). Finally, the NTPsare polymerized by a RNA polymerase (e.g., thermostable RNA polymerase)to form a desired RNA, using a nucleic acid (e.g., DNA) template (FIG.1, reaction 3). The desired synthetic RNA optionally may be purifiedfrom the cell lysate.

Thus, some aspects of the present disclosure provide cell-free methodsof producing (biosynthesizing) ribonucleic acid (RNA), the methodscomprising: (a) incubating at least one cell lysate mixture thatcomprises (i) RNA and (ii) at least one enzymatic activity selected fromthe group consisting of enzymatic activities that depolymerize RNA,thermostable kinase activities, and thermostable RNA polymeraseactivities, under conditions that result in depolymerization of RNA toproduce a cell lysate mixture that comprises nucleoside monophosphates;(b) heating the cell lysate mixture produced in step (a) to atemperature (e.g., 50-80° C.) that inactivates or partially inactivatesendogenous nucleases and phosphatases without completely inactivatingthe thermostable kinase activities and thermostable RNA polymeraseactivities, to produce a cell lysate mixture that comprisesheat-inactivated nucleases and phosphatases; and (c) incubating the celllysate mixture produced in step (b) in the presence of an energy source(e.g., an ATP regeneration system) and a deoxyribonucleic acid (DNA)template (e.g., containing a promoter operably linked to a nucleotidesequence encoding a RNA of interest), under conditions that result inproduction of nucleoside triphosphates and polymerization of thenucleoside triphosphates to produce a cell lysate mixture that comprisesthe RNA of interest.

The cell lysate mixture may comprise a single cell lysate obtained fromcells that comprise RNA and express at least one enzyme (including atleast one fusion enzyme) that acts as a ribonuclease, acts as a kinase,and/or acts as a RNA polymerase. Alternatively, the cell lysate mixturemay comprise at least two (e.g., at least 3, 4, 5, or 6) cell lysates,wherein at least one cell lysate is obtained from cells that compriseRNA, and at least one cell lysate (e.g., at least 2, 3, 4, or 5) isobtained from cells that express at least one enzyme that acts as anuclease, acts as a kinase, and/or acts as a RNA polymerase.

An enzyme or fusion enzyme is considered to “act as a nuclease” if theenzyme of fusion enzyme exhibits nuclease activity (cleaves ordepolymerizes a nucleic acid; e.g., RNase R). An enzyme or fusion enzymeis considered to “act as a kinase” if the enzyme of fusion enzymeexhibits kinase activity (catalyzes the transfer of a phosphate groupfrom one molecule to another molecule; e.g. polyphosphate kinase). Anenzyme or fusion enzyme is considered to “act as a polymerase” if theenzyme of fusion enzyme exhibits polymerase activity (assemblesnucleotides to produce nucleic acids; e.g., RNA polymerase).

In some embodiments, the RNA of step (a) is messenger RNA (mRNA),transfer RNA (tRNA), or ribosomal RNA (rRNA).

In some embodiments, the cell lysate mixture comprises at least oneribonuclease, at least one thermostable kinase, and/or at least one RNApolymerase (e.g., a thermostable RNA polymerase). The use of fusionenzymes is also encompassed by the present disclosure. For example thecell lysate mixture may comprise a fusion of a ribonuclease and akinase, or a fusion of multiple kinases. Other fusion enzymes areencompassed by the present disclosure.

Other aspects of the present disclosure provide engineered cells, celllysates, and cell lysate mixtures comprising at least one nucleosidemonophosphate kinase (e.g., thermostable nucleoside monophosphatekinase), at least one nucleoside diphosphate kinase (e.g., thermostablenucleoside diphosphate kinase), and at least one polyphosphate kinase(e.g., thermostable polyphosphate kinase). The cells, in someembodiments, may also comprise at least one ribonuclease and/or at leastone RNA polymerase (e.g., thermostable RNA polymerase).

In some embodiments, methods of producing (biosynthesizing) RNA comprise(a) lysing cultured cells (e.g., engineered cells) that comprise RNA(e.g., mRNA, tRNA, and/or rRNA), RNase R, thermostable kinases (e.g.,PfPyrH, TthAdk, TthCmk, PfGmk, AaNdk, TePpk, and/or PPK2 (e.g., seeTable 6), and a thermostable T7 RNA polymerase, thereby producing a celllysate, (b) incubating the cell lysate produced in step (a) underconditions that result in depolymerization of RNA to 5′-NMPs, therebyproducing a cell lysate that comprises 5′-NMPs, (c) heating the celllysate produced in step (b) to 60-80° C. to inactivate endogenousnucleases and phosphatases without completely inactivating thethermostable kinases and thermostable RNA polymerase, thereby producinga cell lysate that comprises heat-inactivated nucleases andphosphatases, and (d) incubating the cell lysate produced in step (c) inthe presence of an energy source (e.g., an ATP regeneration systemcomprising polyphosphate) and an engineered nucleic acid (e.g., DNA)template (e.g., containing a promoter operably linked to a nucleotidesequence encoding a RNA of interest), under conditions that result inproduction of nucleoside triphosphates and polymerization of thenucleoside triphosphates to produce the RNA of interest.

In some embodiments, the RNA, RNase R, thermostable kinases, andthermostable T7 RNA polymerase are contained in a single strain ofcultured cells (e.g., engineered cells). In other embodiments, culturedcells (e.g., engineered cells) containing a subset of the aboveactivities/components are lysed, and the lysates combined to generate acell lysate mixture that comprises all the enzymatic activitiesdescribed in step (a) above. In some embodiments, enzymatic activities,in the form of purified enzymes, are added to the lysates described instep (a) above. In some embodiments, lysates and/or purified proteinsare combined before the heat-inactivation step described in step (c)above. In other embodiments, lysates and/or purified proteins arecombined after the heat inactivation step described in step (c) above.

The RNA of interest may be any form of RNA, including single-strandedRNA and double-stranded RNA. For example, the RNA of interest may bemessenger RNA (mRNA), antisense RNA, micro RNA, small interfering RNA(siRNA), or a short hairpin RNA (shRNA). Other RNA interference (RNAi)molecules are encompassed herein.

The details of several embodiments of the invention are set forth in theaccompanying Figures and the Detailed Description. Other features,objects, and advantages of the invention will be apparent from thedescription and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of cell-free RNA production as describedherein. Cells containing RNA from biomass (e.g., endogenous RNA), anuclease, a thermostable kinase, and/or a thermostable RNA polymeraseare lysed (or combined and lysed), and the resulting cell lysate(s)is/are incubated under conditions that result in depolymerization of theRNA. The cell lysate is then heated to inactivate the nuclease and anyendogenous phosphatases (without inactivating the thermostable kinaseand thermostable RNA polymerase). The cell lysate is then incubated inthe presence of an engineered DNA template that encodes a RNA ofinterest, under conditions that result in production of nucleosidetriphosphates and polymerization of the nucleoside triphosphates,thereby producing a RNA of interest (e.g., ssRNA or dsRNA).Alternatively, individual, purified pathway enzymes (e.g., RNApolymerase, such as thermostable RNA polymerase) may be added to thecell lysate following the heat inactivation step. Thus, in someinstances, the engineered cells used to produce the cell lysate do notexpress one or more of the enzymatic activities described above, e.g., anuclease, a thermostable kinase and/or a thermostable RNA polymerase.

FIG. 2A shows a schematic of a polyphosphate-dependent kinase pathwayfor energy generation. FIG. 2B shows a schematic of additional exemplaryenergy conversion pathways for use in the methods and systems of thepresent disclosure. A UMP kinase (e.g., obtained from Pyrococcusfuriosus) and a polyphosphate kinase (e.g., obtained fromThermosynechococcus elongatus, Caldilinea aerophila, Deinococcusgeothermalis, Meiothermus ruber, Meiothermus silvanus, Deinococcusgeothermalis, Anaerolinea thermophila, Chlorobaculum tepidum,Oceanithermus profundus, Roseiflexus castenholzii, Roseiflexus sp., orTruepera radiovctrix) may be used to convert UMP to UDP, and NDP kinases(e.g., encoded by an Aquifex aeolicus ndk gene) and a polyphosphatekinase may be used to convert UDP to UTP. A CMP kinase (e.g., obtainedfrom Thermus thermophilus) and a polyphosphate kinase may be used toconvert CMP to CDP, and NDP kinases and a polyphosphate kinase may beused to convert CDP to CTP. A GMP kinase (e.g., obtained from Thermotogamaritima) and a polyphosphate kinase may be used to convert GMP to GDP,and NDP kinases and a polyphosphate kinase may be used to convert GDP toGTP. An AMP kinase (e.g., obtained from Thermus thermophilus) and apolyphosphate kinase may be used to convert AMP to ADP, and NDP kinases(e.g., encoded by an Aquifex aeolicus ndk gene) and a polyphosphatekinase may be used to convert ADP to ATP. Alternatively, a Class IIIPPK2 enzyme (see, e.g., Table 6) may be used to convert AMP to ATP.

FIG. 3A shows a schematic of an example of a DNA template used for thebiosynthesis of double-stranded RNA. The DNA template, encoded as partof a plasmid, contains a single coding region including of a promoteroperably linked to the coding region of interest and one or moreterminators. Following transcription, the RNA folds into a hairpinstructure through intramolecular nucleotide base pairing. The DNAtemplate, either alone or encoded as part of a plasmid, contains twocomplementary domains (1 and 3), separated by domain 2. FIG. 3B shows aschematic of another example of a DNA template used for biosynthesis ofdouble-stranded RNA. The DNA template contains converging promotersequences on complementary strands. RNA sequences transcribed from eachtemplate strand anneal after transcription. FIG. 3C shows a schematic ofanother example of a DNA template used for biosynthesis ofdouble-stranded RNA. The DNA template, encoded as part of a plasmid,contains converging promoter sequences operably linked to coding regionsof interest on complementary strands, as well as one or more terminatorsequences to prevent read-through transcription. FIG. 3D shows aschematic of another example of a DNA template used for biosynthesis ofdouble-stranded RNA. The DNA template, encoded as part of a plasmid,contains independent cassettes, each including of a promoter operablylinked to the coding region of interest and one or more terminators,driving transcription of complementary sequences, which anneal aftertranscription. FIG. 3E shows a schematic of another example of a DNAtemplate used for biosynthesis of double-stranded RNA. DNA-dependent RNApolymerase is used to produce ssRNA template, and the RNA-dependent RNApolymerase is used to produce double-stranded RNA.

FIG. 4 shows a schematic of another example of a cell-free RNAproduction method of the present disclosure. The process starts with asingle fermentation vessel in which engineered cells are produced usingstandard fermentation techniques. Biomass generated from thefermentation is optionally concentrated by microfiltration (MF) followedby lysis via mechanical homogenization, for example. The lysate is thenpumped into a second fermentation vessel wherein the expressed nucleaseenzymes convert RNA to its monomeric constituents. The entire reactionis heated to inactivate any endogenous phosphatase or nuclease (e.g.,RNase) activities as well as any other exogenous/introduced cellular(e.g., nuclease) activity that would be detrimental to RNA productstability and/or fidelity. Following heat inactivation, polyphosphate isfed to the reaction as a source of high-energy phosphate for thephosphorylation of NMPs to NTPs via a series of thermostable kinases,followed by polymerization to dsRNA. Downstream processing may be usedto increase purity to as much as 99% (e.g., 50%, 60%, 70%, 80%, 90%,95%, 98%, or 99%) dsRNA by weight. For example, processing may be usedto increase purity to 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 70-80%,70-90%, or 70-95%. An exemplary downstream process starts with theaddition of a protein precipitating agent (e.g., ammonium acetate)followed by removal of protein, lipids and some DNA from the productstream by disc stack centrifugation (DSC) or tangential flow filtration(TFF). Ultrafiltration is then implemented to remove salts and reducevolume. Addition of lithium chloride to the product stream leads toprecipitation of the dsRNA product, is subsequently be separated fromthe bulk liquid using disc stack centrifugation, yielding an 80% puritydsRNA product stream. Further chromatographic polishing yields a 99%pure product (Nilsen, T W. Cold Spring Harb Protoc. 2012 Dec. 1; 2012(12)).

FIGS. 5A-5B show a comparison of ribonuclease activities by digestion ofpurified E. coli RNA. (FIG. 5A) Release of acid-soluble nucleotides(mononucleotides and short oligonucleotides) with nuclease treatment wasmost rapid with Benzonase, RNase A, RNase R, and Nuclease P1. (FIG. 5B)LC-MS analysis of reaction products demonstrated NMP release from RNAwith RNase R and Nuclease P1 treatment.

FIG. 6 is a graph showing depolymerization of lysate RNA using exogenousRNase R, with products analyzed by UPLC to specifically identify5′-NMPs. In the absence of RNase R, lysates exhibited endogenous RNaseactivity that led to the slow accumulation of 5′-NMPs (solid dark grayline). Addition of exogenous RNase R led to rapid 5′-NMP release (dasheddark gray line) without affecting rates of 2′ or 3′ NMP accumulation(light gray lines). Thus, overexpression of RNase R accelerates the rateof polymeric RNA to 5′-NMP conversion, reducing the deleterious effectsof phosphatase/nuclease activities present in the extract. Experimentswere performed at a final concentration of 50% lysate.

FIGS. 7A-7C show results of RNase R overexpression in 1 L bioreactorscultures grown in batch phase. (FIG. 7A) SDS-PAGE analysis of proteinexpression in duplicate cultures. Empty vector cultures contained anempty protein expression vector (pETDuet-1). RNase R cultures containedE. coli rnr cloned into pETDuet-1. Samples from induced cultures (+)exhibited strong expression of RNase R (MW 92.9 kDa with C-terminalhexahistidine tag) indicated by arrow at right. (FIG. 7B) Growthkinetics (encompassing pre- and post-induction growth) of Empty Vector(dark gray) and RNase R-expressing strains (light gray) demonstratedthat RNase R overexpression was not deleterious to cell growth. Dashedlines represent exponential curve fits. (FIG. 7C) Overexpressed RNase Rwas active in lysates of batch-grown biomass, releasing acid-solublenucleotides. In the empty vector strain (solid dark gray line), addingexogenous RNase R increased the rate of nucleotide release (dashed darkgray line). In contrast, strains expressing RNase R exhibited rapidnucleotide release upon lysis (solid light gray line). Adding exogenousRNase R did not increase rates of nucleotide release or final nucleotideyields (dashed light gray line). Experiments were performed at a finalconcentration of 50% lysate.

FIG. 8 is a graph describing the effects of chelating Mg²⁺ ondepolymerization rates in high-density lysates. Lysates prepared frombiomass containing Empty Vector (dark gray) were insensitive to EDTA.Lysates with overexpressed RNase R (light gray) exhibited rapid RNAdepolymerization with Mg²⁺ removal, with 8 mM EDTA providing maximumdepolymerization rates. Experiments were performed at a finalconcentration of 90% lysate.

FIGS. 9A-9D show graphs demonstrating stability of exogenousisotopically-labeled “heavy” NMPs (hNMPs) in lysates: (FIG. 9A) hAMP wasrelatively stable in lysates, with 90% remaining after a 1 hourincubation at 37° C. (FIG. 9B) hCMP was degraded in lysates, with 70%remaining after approximately 30 minutes. Addition of 10 mM sodiumorthovanadate (dotted line) (an inhibitor of several phosphatases andkinases) significantly improved stability. (FIG. 9C) hUMP was degradedin lysates, with 70% remaining after approximately 20 minutes. Sodiumphosphate (150 mM) (dashed line) and sodium orthovanadate (dotted line)significantly improved stability. (FIG. 9D) hGMP was degraded inlysates, with 70% remaining after approximately 10 minutes. Sodiumorthovanadate significantly improved stability, with 70% hGMP remainingafter 30 minutes.

FIG. 10 is a graph demonstrating the effect of heat inactivation on thestability of exogenous NTPs in lysates. Lysates were pre-incubated at70° C. before the temperature was lowered to 37° C. and an equimolarmixture of NTPs (ATP, CTP, UTP, and GTP) was added. Pre-incubation timesare listed in the legend at right. Control lysates (not subject to heatinactivation) rapidly consumed NTPs (T=0 min). Increasing pre-incubationtime stabilized NTPs, with 15 minutes at 70° C. eliminating NTPaseactivity (T=15 min).

FIGS. 11A-11B are graphs demonstrating the effect of heat inactivationon the stability of NMPs and dsRNA in lysates. (FIG. 11A) Heatinactivation stabilized NMPs in lysates. Lysates were treated withexogenous RNase R (Lysate+RNase R) (t=0 min-5 min) at 37° C. to releaseNMPs, then heat inactivated (t=5 min to 25 min) at 70° C. Thetemperature was then lowered to 37° C. and the reaction was incubatedfor an additional 60 min. NMPs were largely stable in lysates after heatinactivation. (FIG. 11B) Heat inactivation stabilized reactants andproducts of transcription reactions in lysates. Lysates werepre-incubated for 15 minutes at the indicated temperature, then thetemperature was lowered to 37° C. and transcription reactants wereadded. Heat inactivation at 70° C. and 80° C., but not 60° C. stabilizedsubstrates and products sufficiently to produce a detectabletranscription product similar to the positive control (no lysate).

FIG. 12 is a graph demonstrating temperature-dependent activity of UMPkinase from P. furiosus (PfPyrH), quantified by luciferase assay for ATPconsumption. The specific activity of purified PfPyrH was largelyinsensitive to incubation temperature.

FIG. 13 is a graph demonstrating temperature-dependent activity of AMPkinase from T. thermophilus (TthAdk) compared to Adk from E. coli(EcAdk), measured via luciferase. Purified EcAdk was active attemperatures below 60° C. TthAdk had higher specific activity, with amaximum at 70° C.

FIG. 14 is a graph demonstrating temperature-dependent activity of CMPkinase from T. thermophilus (TthCmk), measured via luciferase. PurifiedTthCmk was relatively insensitive to temperature, with high activityfrom 37-80° C.

FIG. 15 is a graph demonstrating temperature-dependent activity of GMPkinases from E. coli (EcGmk), T. thermophilus (TthGmk), and T. maritima(TmGmk), measured via luciferase. Purified EcGmk (dark gray) was moreactive at lower temperatures, while TthGmk (light gray) and TmGmk(medium gray) were most active at 70° C.

FIG. 16 is a graph of data demonstrating activity of purified NDP kinasefrom A. aeolicus (AaNdk), measured via luciferase. Purified AaNdk washighly active from 37-80° C., using ATP and GDP as substrates, withoptimal activity at 50° C.

FIG. 17 is a graph demonstrating activity of purified polyphosphatekinase 1 (PPK1) enzymes from E. coli (EcPpk), Thermosynechococcuselongatus (TePpk), and Thermus thermophilus (TthPpk) measured vialuciferase. EcPpk was most active at temperatures <60° C., while TePpkwas optimally active at 70° C. TthPpk exhibited relatively low activity.

FIG. 18 is a graph demonstrating activity of commercially-available T7RNA polymerases in buffer using conditions recommended by theirrespective manufacturers at 37° C. ThermoT7 and MegaScript polymerasesexhibited higher specific activity than the NEB polymerase under thetested conditions with duplex DNA template (e.g. in FIG. 3B).

FIG. 19 is a graph comparing T7 RNA polymerase activities in dilutelysates at 37° C. and 50° C. under standardized reaction conditions withduplex DNA template. At 37° C., ThermoT7 exhibited the highest specificactivity. At 50° C., only ThermoT7 had detectable activity, producingover 10 g/L/hr dsRNA.

FIG. 20 is a graph demonstrating activity of ThermoT7 activities inbuffer and high-density heat-inactivated lysates. ThermoT7 activity washighest in lysates clarified by centrifugation after heat-inactivation.Omitting the clarification step led to a 60% decrease in activity,although polymerase activity in unclarified matrix was greater than inbuffer alone.

FIG. 21 is a graph demonstrating tolerance of ThermoT7 to elevatedtemperatures. Pre-incubating ThermoT7 at 50° C. had no effect onsubsequent polymerase activity assayed at 37° C. Pre-incubating at 60°C. and 70° C. led to rapid, irreversible inhibition of enzyme function.

FIG. 22A is an image of an SDS-PAGE gel showing expression andsolubility data for A. thermophila PPK2 in E. coli strain GL16-170. MW:Unstained Protein Standard, Broad Range (New England Biolabs Cat#P7704). −: Pre-induction culture. +: Induced culture at harvest. L:Soluble protein in clarified lysates. A. thermophila PPK2: 33 kDa.

FIG. 22B is a graph showing ATP production of A. thermophila PPK2 inheat-inactivated lysates. Closed circles denote ATP production from ADP.Open circles denote ATP production from AMP. For both substrates, A.thermophila PPK2 produces ATP at rates exceeding 400 mM/hr.

FIG. 23 is an image of an agarose gel demonstrating application ofthermostable Class III PPK2 for energy generation in cell-free dsRNAproduction. Left lanes contain positive controls, demonstrating dsRNAsynthesis from NTPs. Middle lanes contain positive controls,demonstrating dsRNA synthesis from NMPs in nucleotide kinase-expressinglysates using exogenous ATP as an energy source. Right lanes containreactions demonstrating dsRNA synthesis from NMPs and HMP usingnucleotide kinase and C. aerophila Ppk-expressing lysates. In each case,cell-free RNA synthesis reactions are Mn²⁺ independent. Reactionswithout polymerase are included as negative controls, illustratingbackground nucleic acid content of each lysate-containing reaction.

FIG. 24 is an image of an agarose gel demonstrating application ofthermostable Class III PPK2 for energy generation in cell-free dsRNAproduction. Left lanes contain positive controls, demonstrating dsRNAsynthesis from NTPs. Middle lanes contain positive controls,demonstrating dsRNA synthesis from NMPs in nucleotide kinase-expressinglysates using exogenous ATP as an energy source. Right lanes containreactions demonstrating dsRNA synthesis from NMPs and HMP usingnucleotide kinase and C. aerophila Ppk-expressing lysates. With C.aerophila PPK2, dsRNA synthesis proceeds in the absence of AMP kinaseand exogenous ADP.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Provided herein, in some aspects, are methods, compositions, cells,constructs, and systems for the cell-free production (biosynthesis) ofnucleic acid (e.g., RNA or DNA). In some embodiments, a single type oforganism (e.g., a population of bacterial cells) is engineered toexpress at least one nuclease, at least one thermostable kinases and atleast one thermostable polymerase (e.g., RNA or DNA polymerase). Theengineered cells are grown (cultured) under conditions that result inenzyme expression. In some embodiments, the engineered cells may begrown to a desired cell density, and then expression of certain enzymesmay be induced (activated). Thus, transcription of certain enzymes maybe under the control of an inducible promoter. The cells (e.g.,engineered and/or non-engineered cells) are then lysed (e.g.,mechanically, chemically, or enzymatically disrupted) to produce a celllysate that comprises the enzymatic activities required for cell-freeproduction of RNA (e.g., ssRNA or dsRNA). In some embodiments, cellscontaining polymeric RNA (e.g., mRNA, tRNA, and/or rRNA) are mixed withthe engineered cells containing pathway enzymes prior to the cell lysisstep. In other embodiments, cell lysate(s) obtained from cellscontaining polymeric RNA is combined (mixed) with cell lysate(s)obtained from engineered cells containing pathway enzymes. In yet otherembodiments, one or more purified pathway enzymes are combined (mixed)with cell lysate(s) obtained from engineered cells. “Pathway enzymes”are enzymes required to biosynthesize the RNA of interest (e.g.,starting from polymeric RNA).

To synthesize RNA, the cell lysate (or cell lysate mixture) is incubatedunder conditions that result in nuclease-mediated (e.g., RNase-mediated)depolymerization of the host-derived (endogenous) RNA to a desired yieldof 5′-nucleoside monophoshates (NMPs, or nucleoside monophosphates). Thecell lysate (or cell lysate mixture) is then heated, in someembodiments, to inactivate the majority of host-derived enzymes,including phosphatases and nucleases (e.g., RNases), as well as anyexogenous nuclease(s) previously added to the cell lysate to facilitatedepolymerization of the host-derived RNA. Following the heatinactivation step, the cell lysate is incubated under conditions thatresult in phosphorylation of the NMPs to NTPs (nucleoside triphosphates)by thermostable kinases (e.g., thermostable nucleoside monophosphatekinases and nucleoside diphosphate kinases) using, for example,thermostable polyphosphate kinase and the addition of polyphosphate asthe energy source. The resulting NTPs are subsequently polymerized toRNA by a RNA polymerase (e.g., thermostable RNA polymerase) using anengineered template (e.g., DNA template) present in the lysates (e.g.,either expressed by the engineered cells and included as a cellularcomponent of the cell lysate, or later added to the cell lysate).

Cell-Free Production

“Cell-free production” is the use of biological processes for thesynthesis of a biomolecule or chemical compound without using livingcells. The cells are lysed and unpurified (crude) portions orpartially-purified portions, both containing enzymes, are used for theproduction of a desired product. Purified enzymes may be added to celllysates, in some embodiments. As an example, cells are cultured,harvested, and lysed by high-pressure homogenization or other cell lysismethod (e.g., chemical cell lysis). The cell-free reaction may beconducted in a batch or fed-batch mode. In some instances, the enzymaticpathways fill the working volume of the reactor and may be more dilutethan the intracellular environment. Yet substantially all of thecellular catalysts are provided, including catalysts that are membraneassociated. The inner membrane is fragmented during cell lysis, and thefragments of these membranes may form membrane vesicles. See, e.g.,Swartz, AIChE Journal, 2012, 58(1), 5-13, incorporated herein byreference.

Cell-free methods, compositions, and systems of the present disclosureutilize cell lysates (e.g., crude or partially purified cell lysates),discussed in greater detail herein. Cell lysates prepared, for example,by mechanical means (e.g., shearing or crushing), are distinct fromchemically-permeabilized cells. As discussed above, in some embodiments,during cell lysis (e.g., mechanical cell lysis), the inner cell membraneis fragmented such that inverted membrane vesicles are formed in thecell lysates. Such inverted membrane vesicles are not produced throughchemical cell permeabilization methods. Cells that are lysed (e.g., atleast 75%, 80%, 85%, 90%, or 95%) are no longer intact. Thus,permeabilized cells, which are intact cells containing perforations(small holes) are not considered lysed cells.

While the methods provided herein are generally cell-free and use celllysates, in some embodiments, it may be advantageous, at least for somesteps of the methods, to use permeabilized cells. Thus, the presentdisclosure does not exclude the use of permeabilized cells in at leastone step of the RNA production methods.

It should be understood that while many of the embodiments describedherein refer to “lysing cultured cells” that comprise particularenzymes, the phrase is intended to encompass lysing a clonal populationof cells obtained from a single culture (e.g., containing all theenzymes needed to synthesize RNA) as well as lysing more than one clonalpopulation of cells, each obtained from different cell cultures (e.g.,each containing one or more enzymes needed to synthesize RNA and/or thepolymeric RNA substrate). For example, in some embodiments, a populationof cells (e.g., engineered cells) expressing one thermostable kinase maybe cultured together and used to produce one cell lysate, and anotherpopulation of cells (e.g., engineered cells) expressing a differentthermostable kinase may be cultured together and used to produce anothercell lysate. These two cell lysates, each comprising a differentthermostable kinase, may then be combined for use in a RNA biosynthesismethod of the present disclosure.

Depolymerization of Ribonucleic Acid to Nucleoside Monophosphates

The present disclosure is based on the conversion of RNA from biomass(e.g., endogenous cellular RNA) to desired synthetic RNA using a celllysate through a cell-free process involving a series of enzymaticreactions. First, RNA (e.g., endogenous RNA) present in a cell lysate,derived from host cells, is converted to its constituent monomers bynucleases. RNA from biomass (e.g., endogenous RNA) typically includesribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), otherRNAs, or a combination thereof. Depolymerization or degradation of RNAresults in a pool of 5′-nucleoside monophosphates (5′-NMPs), alsoreferred to simply as “monomers.” These monomers, which are converted tonucleoside diphosphates, which are converted to nucleosidetriphosphates, are used as starting material for the downstreampolymerization/synthesis of a RNA of interest. In some embodiments, theRNA of interest is ssRNA (e.g., mRNA). In some embodiments, the RNA ofinterest is dsRNA.

The amount of RNA (e.g., endogenous RNA) required to synthesize a RNA ofinterest may vary, depending on, for example, the desired length andyield of the RNA of interest as well as the nucleotide composition ofthe RNA relative to the nucleotide composition of the RNA (e.g.,endogenous RNA) of the cell (e.g., E. coli cell). Typically, for abacterial cell, for example, RNA (e.g., endogenous RNA) content rangesfrom 5-50% of the total cell mass. The mass of the starting material canbe calculated, for example, using the following equation: (kilogram (kg)of RNA/kilogram of dry cell weight)×100%.

Endogenous RNA may be depolymerized or degraded into its constituentmonomers by chemical or enzymatic means. Chemical hydrolysis of RNA,however, typically produces 2′- and 3′-NMPs, which cannot be polymerizedinto RNA. Thus, the methods, compositions, and systems as providedherein primarily use enzymes for the depolymerization of endogenous RNA.An “enzyme that depolymerizes RNA” catalyzes the hydrolysis of thephosphodiester bonds between two nucleotides in a RNA. Thus, “an enzymethat depolymerizes RNA” converts RNA (polymeric RNA) into its monomericform—nucleoside monophosphates (NMPs). Depending on the enzyme,enzymatic depolymerization of RNA may yield 3′-NMPs, 5′-NMPs or acombination of 3′-NMPs and 5′-NMPs. Because it is not possible topolymerize 3′-NTPs (converted from 3′-NDPs, which are converted from3′-NMPs), enzymes (e.g., RNase R) that yield 5′-NMPs (which are thenconverted to 5′-NDPs, and then 5′-NTPs) are preferred. In someembodiments, enzymes that yield 3′-NMPs are removed from the genomic DNAof the engineered cell to increase efficiency of RNA production. In someembodiments, the enzyme used for RNA depolymerization is RNase R. Insome embodiments, the concentration of RNase R used is 0.1-1.0 mg/mL(e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. or 1.0 mg/mL). Insome embodiments, the concentration of RNase R used is 0.4-0.6 mg/mL. Insome embodiments, the concentration of RNase R used is 0.5 mg/mL. Insome embodiments, the concentration of RNase R used is greater than 1.0mg/mL.

Examples of enzymes that depolymerize RNA include, without limitation,nucleases, including ribonucleases (RNases, e.g., RNase R) andphosphodiesterases. Nucleases catalyze the degradation of nucleic acidinto smaller components (e.g., monomers, also referred to as nucleosidemonophosphates, or oligonucleotides). Phosphodiesterases catalyzedegradation of phosphodiester bonds. These enzymes that depolymerize RNAmay be encoded by full length genes or by gene fusions (e.g., DNA thatincludes at least two different genes (or fragments of genes) encodingat two different enzymatic activities).

RNase functions in cells to regulate RNA maturation and turn over. EachRNase has a specific substrate preferences—dsRNA or ssRNA. Thus, in someembodiments, a combination of different RNases, or a combination ofdifferent nucleases, generally, may be used to depolymerizebiomass-derived polymeric RNA (e.g., endogenous RNA). For example, 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 different nucleases may beused in combination to depolymerize RNA. In some embodiments, at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 different nucleases may beused in combination to depolymerize RNA. Non-limiting examples ofnucleases for use as provided herein are included in Table 1. In someembodiments, the nuclease used is RNase R.

TABLE 1 Enzymes that Depolymerize Ribonucleic Acid Nuclease HostOrganism(s) EC # UniProt Reference Nuclease Penicillium citrum 3.1.30.1P24289 1, 2, 3 P1 (P1 Nuclease) RNase II Escherichia coli 3.1.13.1P30850 4, 5 RNase III Escherichia coli 3.1.26.3 P0A7Y0 6, 7, 8 RNase RPseudomonas putida or 3.1.13.— R9V9M9 9 Escherichia coli P21499 RNase JIBacillus subtilis 3.1.4.1 Q45493 10, 11 NucA Serratia marcescens3.1.30.2 P13717 12, 13, 14 RNase T Escherichia coli 3.1.27.3 P30014 15,16, 17 RNase E Escherichia coli 3.1.26.12 P21513 18, 19

Enzymes that depolymerize RNA (e.g., RNases) may be endogenous to a hostcell (host-derived), or they may be encoded by engineered nucleic acidsexogenously introduced into a host cell (e.g., on an episomal vector orintegrated into the genome of the host cell).

In some embodiments, engineered nucleic acids encoding enzymes thatdepolymerize RNA are operably linked to an inducible promoter. Thus, insome embodiments, expression of an engineered nucleic acid encoding anenzyme that depolymerizes RNA is temporally or spatially regulated. Forexample, nucleic acids may be engineered to encode enzymes (e.g.,RNases) that are relocated to or are sequestered in the periplasm of ahost cell so that activity of the enzyme does not interfere with cellgrowth or other metabolic processes. Upon cell lysis, the relocatedenzyme is released from the periplasm, brought into contact with theendogenous RNA, and depolymerizes the RNA into monomeric form. See,e.g., International Publication No. WO 2011/140516, published Nov. 10,2011, incorporated herein by reference.

“Conditions that result in depolymerization of RNA” are known in the artor may be determined by one of ordinary skill in the art, taking intoconsideration, for example, optimal conditions for nuclease (e.g.,RNase) activity, including pH, temperature, length of time, and saltconcentration of the cell lysate as well as any exogenous cofactors.Examples including those described previously (see, e.g., Wong, C. H. etal. J. Am. Chem. Soc., 105: 115-117, 1983), EP1587947B1, Cheng Z F,Deutscher M P. J. Biol Chem. 277:21624-21629, 2002).

In some embodiments, metal ions (e.g., Mg²⁺) are depleted from thedepolymerization reaction. In some embodiments, the concentration ofmetal ion (e.g., Mg²⁺) is 8 mM or less (e.g., less than 8 mM, less than7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM,less than 2 mM, less than 1 mM, less than 0.5 mM). In some embodiments,the concentration of metal ion (e.g., Mg²⁺) is 0.1 mM-8 mM, 0.1 mM-7 mM,or 0.1 mM-5 mM.

The pH of a cell lysate during a RNA depolymerization reaction may havea value of 3.0 to 8.0. In some embodiments, the pH value of a celllysate is 3.0-8.0, 4.0-8.0, 5.0-8.0, 6.0-8.0, 7.0-8.0, 3.0-7.0, 4.0-7.0,5.0-7.0, 6.0-7.0, 3.0-6.0, 4.0-6.0, 5.0-6.0, 3.0-5.0, 3.0-4.0, or4.0-5.0. In some embodiments, the pH value of a cell lysate is 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, thepH value of a cell lysate is 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. The pH ofa cell lysate may be adjusted, as needed.

The temperature of a cell lysate during a RNA depolymerization reactionmay be 15° C. to 70° C. In some embodiments, the temperature of a celllysate during a RNA depolymerization reaction is 15-60° C., 15-50° C.,15-40° C., 15-30° C., 25-70° C., 25-60° C., 25-50° C., 25-40° C., 30-70°C., 30-60° C., or 30-50° C. In some embodiments, the temperature of acell lysate during a RNA depolymerization reaction is 37° C. In someembodiments, the temperature of a cell lysate during a RNAdepolymerization reaction is 15° C., 25° C., 32° C., 37° C., 40° C., 42°C., 45° C., 50° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61°C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., or70° C.

A cell lysate during a RNA depolymerization reaction may be incubatedfor 5 minutes (min) to 72 hours (hrs). In some embodiments, a celllysate during a RNA depolymerization reaction is incubated for 5-10 min,5-15 min, 5-20 min, 5-30 min, or 5 min-48 hrs. For example, a celllysate during a RNA depolymerization reaction may be incubated for 5min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 1 hr, 2 hrs, 3 hrs,4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 18hrs, 24 hrs, 30 hrs, 36 hrs, 42 hours, or 48 hours. In some embodiments,a cell lysate during a RNA depolymerization reaction is incubated for 24hours at a temperature of 37° C. In some embodiments, a cell lysateduring a RNA depolymerization reaction is incubated for 5-10 min at atemperature of 37° C. In some embodiments, a cell lysate during a RNAdepolymerization reaction has a pH of 7.0 and is incubated for 15minutes at a temperature of 37° C. In some embodiments, a cell lysateduring a RNA depolymerization reaction may be incubated under conditionsthat result in greater than 65% conversion of RNA to 5′-NMPs. In someembodiments, RNA is converted to 5′-NMPs at a rate of (or at least) 50mM/hr, 100 mM/hr or 200 mM/hr.

In some embodiments, salt is added to a cell lysate, for example, toprevent enzyme aggregation. For example, sodium chloride, potassiumchloride, sodium acetate, potassium acetate, or a combination thereof,may be added to a cell lysate. The concentration of salt in a celllysate during a RNA depolymerization reaction may be 5 mM to 1 M. Insome embodiments, the concentration of salt in a cell lysate during aRNA depolymerization reaction 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM,100 mM, 150 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M. In someembodiments, the cell lysate comprises a mixture that includes 40-60 mMpotassium phosphate, 1-5 mM MnCl₂, and/or 10-50 mM MgCl₂ (e.g., 20 mMMgCl₂).

In some embodiments, buffer is added to a cell lysate, for example, toachieve a particular pH value and/or salt concentration. Examples ofbuffers include, without limitation, phosphate buffer, Tris buffer, MOPSbuffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MESbuffer, histidine buffer, PIPES buffer, bis-tris buffer, andethanolamine buffer.

Depolymerization of RNA results in the production of 5′-NMP, including5′-AMP, 5′-UMP, 5′-CMP, and 5′-GMP. While the NMP may be present in thecell lysate at relatively equimolar amounts, depolymerization of RNAdoes not result in any predetermined ratio of NMPs.

In some embodiments, 50-98% of the endogenous RNA in a cell upon lysisis converted to (depolymerized to) 5′-NMPs. For example, 50-95%, 50-90%,50-85%, 50-80%, 75-98%, 75-95%, 75-90%, 75-85% or 75-80% RNA isconverted to (depolymerized to) 5′-NMPs. In some embodiments, 65-70% ofthe endogenous RNA in a cell upon lysis is converted to (depolymerizedto) 5′-NMPs. Lower yields are also acceptable.

Elimination of Futile Cycles

Following conversion of RNA from biomass (e.g., endogenous RNA) to itsmonomeric constituents by endogenous and/or exogenous nucleases, theretypically remains in the cell lysate several enzymes, includingnucleases and phosphatases, which may have deleterious effects on RNAbiosynthesis. For example, Escherichia coli has numerous phosphatases,many of which dephosphorylate NTPs, NDPs, and NMPs. Dephosphorylation ofNMPs following RNA depolymerization results in the accumulation of thenon-phosphorylated nucleosides and a loss of usable NMP substrate, thusreducing synthetic RNA yield. Dephosphorylation of NMPs, NDPs, or NTPsfollowing RNA depolymerization results in futile energy cycles (energycycles that produce a low yield of synthetic RNA) during which NMPs arephosphorylated to NDPs and NTPs, which in turn are dephosphorylated backto their NMP or nucleoside starting point. Futile cycles reduce theyield of RNA product per unit energy input (e.g., polyphosphate, ATP, orother sources of high energy phosphate). In some embodiments, theenzymatic activities are eliminated by removal from the host genome. Insome embodiments, the enzymatic activities are eliminated by heatinactivation. In some embodiments, the enzymatic activities areeliminated by protease targeting. In some embodiments the enzymaticactivities are eliminated through the use of chemical inhibitors. Acombination of any of the foregoing approaches may also be used.

Enzymes deleterious to the biosynthesis of RNA, as provided herein, maybe deleted from the host cell genome during the process of engineeringthe host cell, provided the enzymes are not essential for host cell(e.g., bacterial cell) survival and/or growth. Deletion of enzymes orenzyme activities may be achieved, for example, by deleting or modifyingin the host cell genome a gene encoding the essential enzyme. An enzymeis “essential for host cell survival” if the enzyme is necessary for thesurvival of the host cell. That is, if a host cell cannot survivewithout expression and/or activity of a particular enzyme, then thatenzyme is considered essential for host cell survival. Similarly, anenzyme is “essential for host cell growth” if the enzyme is necessaryfor the growth of the host cell. That is, if a host cell cannot divideand/or grow without expression and/or activity of a particular enzyme,then that enzyme is considered essential for host cell growth.

If enzymes deleterious to the biosynthesis of RNA are essential for hostcell survival and/or growth, then it may not be possible to delete ormodify the genes encoding the enzymes. In such instances, the enzymesmay be heat inactivated. “Heat inactivation” refers to the process ofheating a cell lysate to a temperature sufficient to inactivate (or atleast partially inactivate) endogenous nucleases and phosphatases.Generally, the process of heat inactivation involves denaturation of(unfolding of) the deleterious enzyme. The temperature at whichendogenous cellular proteins denature varies among organisms. In E.coli, for example, endogenous cellular enzymes generally denature attemperatures above 41° C. The denaturation temperature may be higher orlower than 41° C. for other organisms. Enzymes of a cell lysate, asprovide here, may be heat inactivated at a temperature of 40° C.-95° C.,or higher. In some embodiments, enzymes of a cell lysate may be heatinactivated at a temperature of 40-90° C., 40-80° C., 40-70° C., 40-60°C., 40-50° C., 50-80° C., 50-70° C., 50-60° C., 60-80° C., 60-70° C., or70-80° C. For example, enzymes of a cell lysate may be heat inactivatedat a temperature of 40° C., 42° C., 45° C., 50° C., 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95° C. In someembodiments, enzymes of a cell lysate may be heat inactivated at atemperature of 50-80° C. In some embodiments, enzymes of a cell lysatemay be heat inactivated at a temperature of 70° C. In some embodiments,enzymes of a cell lysate may be heat inactivated at a temperature of 60°C. It may also be possible to introduce chemical inhibitors ofdeleterious enzymes. Such inhibitors may include, but are not limitedto, sodium orthovanadate (inhibitor of protein phosphotyrosylphosphatases), sodium fluoride (inhibitor of phosphoseryl andphosphothreonyl phosphatases), sodium pyrophosphate (phosphataseinhibitor), sodium phosphate, and/or potassium phosphate.

The period of time during which a cell lysate is incubated at elevatedtemperatures to achieve heat inactivation of endogenous enzymes mayvary, depending, for example, on the volume of the cell lysate and theorganism from which the cell lysate was prepared. In some embodiments, acell lysate is incubated at a temperature of 35° C.-80° C. for 2 minutes(min) to 48 hours (hr). For example, a cell lysate may be incubated at atemperature of 35° C.-80° C. for 2 min, 4 min, 5 min, 10 min, 15 min, 30min, 45 min, or 1 hr. In some embodiments, a cell lysate is incubated ata temperature of 35° C.-80° C. for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 42, or 48 hr.

In some embodiments, enzymes are heat inactivated at a temperature of60-80° C. for 10-20 min. In some embodiments, enzymes are heatinactivated at a temperature of 70° C. for 15 min.

In some embodiments, enzymes that depolymerize endogenous RNA compriseone or more modifications (e.g., mutations) that render the enzymes moresensitive to heat. These enzymes are referred to as “heat-sensitiveenzymes.” Heat-sensitive enzymes denature and become inactivated attemperatures lower than that of their wild-type counterparts, and/or theperiod of time required to reduce the activity of the heat-sensitiveenzymes is shorter than that of their wild-type counterparts.

It should be understood that enzymes that are heat inactivated may, insome instances, retain some degree of activity. For example, theactivity level of a heat-inactivated enzyme may be less than 50% of theactivity level of the same enzyme that has not been heat inactivated. Insome embodiments, the activity level of a heat-inactivated enzyme isless than 40%, less than 30%, less than 20%, less than 10%, less than5%, less than 1%, or less than 0.1% of the activity level of the sameenzyme that has not been heat inactivated.

Thus, an enzyme's activity may be completely eliminated or reduced. Anenzyme is considered completely inactive if the denatured (heatinactivated) form of the enzyme no longer catalyzes a reaction catalyzedby the enzyme in its native form. A heat-inactivated, denatured enzymeis considered “inactivated” when activity of the heat-inactivated enzymeis reduced by at least 50% relative to activity of the enzyme that isnot heated (e.g., in its native environment). In some embodiments,activity of a heat-inactivated enzyme is reduced by 50-100% relative tothe activity of the enzyme that is not heated. For example, activity ofa heat-inactivated enzyme is reduced by 50-90%, 50-85%, 50-80%, 50-75%,50-70%, 50-65%, 50-60%, or 50-55% relative to activity of the enzymethat is not heated. In some embodiments, the activity of aheat-inactivated enzyme is reduced by 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% relative tothe activity of the enzyme that is not heated.

Examples of enzymes that may be heat inactivated, or deleted from thegenome of a host cell, include, without limitation, nucleases (e.g.,RNase III, RNase I, RNase R, PNPase, RNase II, and RNase T),phosphatases (e.g., nucleoside monophosphatase, nucleosidediphosphatase, nucleoside triphosphatase), and other enzymes thatdepolymerize RNA or dephosphorylate nucleotides. Enzymes thatdepolymerize RNA include any enzyme that is able to cleave, partiallyhydrolyze, or completely hydrolyze a RNA molecule. Table 2 provides alist of non-limiting examples of nucleases that may be heat inactivatedor, in some instances, deleted from an engineered host cell. Table 3provides a list of non-limiting examples of phosphatases that may beheat inactivated or, in some instances, deleted from an engineered hostcell. Heat inactivation of these and other nucleases and phosphatases isencompassed by the present disclosure.

TABLE 2 Examples of Nucleases Nuclease Gene Function EC # UniprotReference RNase III rnc Cleaves dsRNA, rRNA and some mRNA 3.1.26.3P0A7Y0 6, 7, 8 RNase I rna General ribonuclease, broad substrate3.1.27.6 P21338 20 specificity. Localizes to periplasm. RNase R rnrCleaves some dsRNA, poly-A mRNA, 3.1.13.— P21499 4, 21 mRNA and rRNAPNPase pnp General mRNA degradation, tRNA 3.1.13.1 P05055 22, 23, 24maturation and degradation. RNase II rnb Exonuclease. Plays a role intRNA 3.1.13.1 P30850 4, 5 processing RNase T rnt Processing of tRNAs,rRNA and other 3.1.13.— P30014 15, 16, 17 stable RNAs. Capable ofdegrading ssDNA and ssRNA. RNase E rne Processes rRNA, tRNA and otherRNAs. 3.1.26.12 P21513 18, 19 Associates with the “degradasome”

TABLE 3 Examples of Phosphatases. Phosphatase Class Host EC # ExamplesUniprot Reference NMP/NDP E. coli 3.1.3.5 AphA P0AE22 25, 26 phosphatase3.6.1.6 PhoA P00634 27, 28 UmpG P0A840 29 YrfG P64636 30 UshA P07024 31,32 UmpH P0AF24 33 NTP phosphatase E. coli 3.6.1.15 AppA P07102 34, 35RavA P31473 36 NTP E. coli 3.6.1.19 YhdE P25536 37 phosphohydrolase MazGP0AEY3 38

E. coli RNase III preferentially cleaves dsRNA as well as somesingle-stranded mRNA molecules. The presence of RNase III in cell lysatemay limit the accumulation of high concentrations of synthetic RNA(e.g., dsRNA), because the synthetic RNA is readily cleaved. NeitherRNase III nor the gene encoding RNase III, mc, is essential for cellviability, thus, in some embodiments, mc is deleted or mutated inengineered host cells. In other embodiments, RNase III is heatinactivated following depolymerization of endogenous RNA.

E. coli RNase I localizes to the periplasmic space in intact cells andcatalyzes depolymerization of a wide range of RNA molecules, includingrRNA, mRNA, and tRNA. Under physiological conditions the periplasmiclocalization of this enzyme means that the enzyme has little impact onRNA stability within the cell; however, mixing of the periplasm andcytoplasm in cell lysates permits RNase I access to cellular RNA. Thepresence of RNaseI in a cell lysate may reduce the yield of syntheticRNA through RNA degradation. Neither RNase I nor the gene encoding RNaseI, ma, is essential for cell viability, thus, in some embodiments, ma isdeleted or mutated in engineered host cells. In other embodiments, RNaseI is heat inactivated following depolymerization of endogenous RNA.

E. coli RNase R and RNase T catalyze the depolymerization of dsRNA,rRNA, tRNA, and mRNA, as well as small unstructured RNA molecules.Neither the enzymes nor the genes encoding the enzymes, rnr and rnt,respectively, are essential for cell viability, thus, in someembodiments, rnr and/or rnt are deleted or mutated in engineered hostcells (e.g., E. coli host cells). In other embodiments, RNase R and/orRNase T are heat inactivated following the depolymerization ofendogenous RNA.

E. coli RNase E and PNPase are components of the degradasome, which isresponsible for mRNA turnover in cells. RNase E is thought to functiontogether with PNPase and RNase II to turn over cellular mRNA pools.Disruption of the gene encoding RNase E, me, is lethal in E. coli. Thus,in some embodiments, RNase E is heat inactivated followingdepolymerization of endogenous RNA. Neither PNPase nor the gene encodingPNPase, pnp, is essential for cell viability, thus, in some embodiments,pnp is deleted or mutated in engineered host cells (e.g., E. coli hostcells). In other embodiments, PNPase is heat inactivated followingdepolymerization of endogenous RNA.

E. coli RNase II depolymerizes both mRNA and tRNA in a 3′ 4 5′direction. Neither RNase II nor the gene encoding RNase II, rnb, isessential for cell viability, thus, in some embodiments, rnb is deletedor mutated in engineered host cells. In other embodiments, RNase II isheat inactivated following depolymerization of endogenous RNA.

While neither pnp nor rnb is essential to host cell survival, disruptionof both simultaneously may be lethal. Thus, in some embodiments, bothPNPase and RNase II are heat inactivated.

Phosphorylation of Nucleoside Monophosphates to Nucleoside Triphosphates

Following conversion of endogenous RNA to its monomeric form, andfollowing heat inactivation of endogenous nucleases and phosphatases,the resulting nucleoside monophosphates (NMPs) in a cell lysate arephosphorylated before they are polymerized to form a desired syntheticRNA, such as a double-stranded RNA or single-stranded RNA (e.g., mRNA orantisense RNA). This process is highly energy dependent, thus, thisprocess requires an energy source. The phosphates are typically donatedfrom a high-energy phosphate source, such as, for example,phosphoenolpyruvate, ATP, or polyphosphate.

In some embodiments, the energy source is ATP that is directly added toa cell lysate. In other embodiments, the energy source is provided usingan ATP regeneration system. For example, polyphosphate and polyphosphatekinase may be used to produce ATP. Other examples included the use ofacetyl-phosphate and acetate kinase to produce ATP; phospho-creatine andcreatine kinase to produce ATP; and phosphoenolpyruvate and pyruvatekinase to produce ATP. Other ATP (or other energy) regeneration systemsmay be used. In some embodiments, at least one component of the energysource is added to a cell lysate or cell lysate mixture. A “component”of an energy source includes the substrate(s) and enzyme(s) required toproduce energy (e.g., ATP). Non-limiting examples of these componentsinclude polyphosphate, polyphosphate kinase, acetyl-phosphate, acetatekinase, phospho-creatine, creatine kinase, phosphoenolpyruvate, andpyruvate kinase.

A kinase is an enzyme that catalyzes the transfer of phosphate groupsfrom high-energy, phosphate-donating molecules, such as ATP, to specificsubstrates/molecules. This process is referred to as phosphorylation,where the substrate gains a phosphate group and the high-energy ATPmolecule donates a phosphate group. This transesterification produces aphosphorylated substrate and ADP. The kinases of the present disclosure,in some embodiments, convert the NMPs to NDPs and NDPs to NTPs.

In some embodiments, a kinase is a nucleoside monophosphate kinase,which catalyzes the transfer of a high-energy phosphate from ATP to anNMP, resulting in ADP and NDP. Non-limiting examples of nucleosidemonophosphate kinases are provided in Tables 4 and 5. As discussedbelow, thermostable variants of the enzymes listed in Tables 4 and 5 areencompassed by the present disclosure. In some embodiments, a celllysate comprises one or more (or all) of the following four nucleosidemonophosphate kinases: thermostable uridylate kinase, thermostablecytidylate kinase, thermostable guanylate kinase and thermostableadenylate kinase. In some embodiments, UMP kinase is obtained fromPyrococcus furiosus (e.g., SEQ ID NO:3 or a variant comprising an aminoacid sequence that is at least 70% identical to the amino acid sequenceidentified by SEQ ID NO:3). In some embodiments, CMP kinase is obtainedfrom Thermus thermophilus (e.g., SEQ ID NO:4 or a variant comprising anamino acid sequence that is at least 70% identical to the amino acidsequence identified by SEQ ID NO:4). In some embodiments, GMP kinase isobtained from Thermotoga maritima (e.g., SEQ ID NO:5 or a variantcomprising an amino acid sequence that is at least 70% identical to theamino acid sequence identified by SEQ ID NO:5). In some embodiments, AMPkinase is obtained from Thermus thermophilus (e.g., SEQ ID NO:6 or avariant comprising an amino acid sequence that is at least 70% identicalto the amino acid sequence identified by SEQ ID NO:6).

Thus, in some embodiments, a NMP kinase has an amino acid sequenceidentified by the amino acid sequence of any one of SEQ ID NO: 3-6. Insome embodiments, the NMP kinase has an amino acid sequence that is atleast 70% identical to the amino acid sequence of any one of SEQ ID NO:3-6. For example, the NMP kinase may have an amino acid sequence that isat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to theamino acid sequence identified by any one of SEQ ID NO: 3-6.

It should be understood that the present disclosure encompasses the useof any one or more of the enzymes described herein as well as variantsof the enzymes (e.g., “PPK2 variants”). Variant enzymes may share acertain degree of sequence identity with the reference enzyme. The term“identity” refers to a relationship between the sequences of two or morepolypeptides or polynucleotides, as determined by comparing thesequences. Identity measures the percent of identical matches betweenthe smaller of two or more sequences with gap alignments (if any)addressed by a particular mathematical model or computer program (e.g.,“algorithms”). Identity of related molecules can be readily calculatedby known methods. “Percent (%) identity” as it applies to amino acid ornucleic acid sequences is defined as the percentage of residues (aminoacid residues or nucleic acid residues) in the candidate amino acid ornucleic acid sequence that are identical with the residues in the aminoacid sequence or nucleic acid sequence of a second sequence afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent identity. Identity depends on a calculation ofpercent identity but may differ in value due to gaps and penaltiesintroduced in the calculation. Variants of a particular sequence mayhave at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% but less than 100% sequence identity to that particularreference sequence, as determined by sequence alignment programs andparameters described herein and known to those skilled in the art.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. Techniques for determining identity are codified in publiclyavailable computer programs. Exemplary computer software to determinehomology between two sequences include, but are not limited to, GCGprogram package (Devereux, J. et al. Nucleic Acids Research, 12(1): 387,1984), the BLAST suite (Altschul, S. F. et al. Nucleic Acids Res. 25:3389, 1997), and FASTA (Altschul, S. F. et al. J. Molec. Biol. 215: 403,1990). Other techniques include: the Smith-Waterman algorithm (Smith, T.F. et al. J. Mol. Biol. 147: 195, 1981; the Needleman-Wunsch algorithm(Needleman, S. B. et al. J. Mol. Biol. 48: 443, 1970; and the FastOptimal Global Sequence Alignment Algorithm (FOGSAA) (Chakraborty, A. etal. Sci Rep. 3: 1746, 2013).

TABLE 4 Examples of Nucleoside Monophosphate Kinases Enz. Name HostOrganism EC # Reaction Uniprot Ref. PyrH E. coli 2.7.4.22 UMP + ATP →P0A7E9 39, 40 T. thermophilus UDP + ADP P43891 41 P. furiosus Q8U122 42,43 Cmk E. coli 2.7.4.25 CMP + ATP → P0A6I0 44, 45 T. thermophilus CDP +ADP Q5SL35 41 P. furiosus Q8U2L4 46 Gmk E. coli 2.7.4.8 GMP + ATP →P60546 47, 48 T. thermophilus GDP + ADP Q5SI18 41 T. maritima Q9X215 49Adk E. coli 2.7.4.3 AMP + ATP → P69441 50, 51 T. thermophilus 2 ADPQ72I25 52, 53 P. furiosus Q8U207 46

In some embodiments, a kinase is a nucleoside diphosphate kinase, whichtransfers a phosphoryl group to NDP, resulting in NTP. The donor of thephosphoryl group may be, without limitation, ATP, polyphosphate polymer,or phosphoenolpyruvate. Non-limiting examples of kinases that convertNDP to NTP include nucleoside diphosphate kinase, polyphosphate kinase,and pyruvate kinase. As discussed below, thermostable variants of theforegoing enzymes are encompassed by the present disclosure. In someembodiments, the NDP kinase(s) is/are obtained from Aquifex aeolicus(e.g., SEQ ID NO: 9 or a variant comprising an amino acid sequence thatis at least 70% identical to the amino acid sequence identified by SEQID NO:9). In some embodiments, the NDP kinase has an amino acid sequencethat is at least 70% identical to the amino acid sequence identified bySEQ ID NO: 9. For example, the NDP kinase may have an amino acidsequence that is at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the amino acid sequence identified by SEQ ID NO: 9.

Phosphorylation of NMPs to NTPs occurs, in some embodiments, through thepolyphosphate-dependent kinase pathway (FIGS. 2A and 2B), wherehigh-energy phosphate is transferred from polyphosphate to ADP via apolyphosphate kinase (PPK). In some embodiments, the polyphosphatekinase belongs to the polyphosphate kinase 1 (PPK1) family, whichtransfers high-energy phosphate from polyphosphate to ADP to form ATP.This ATP is subsequently used by NMP kinases (e.g., AMP kinase, UMPkinase, GMP kinase, and CMP kinase) to convert NMPs to their cognateribonucleotide diphosphates (NDPs). Furthermore, ATP is subsequentlyused by nucleotide diphosphate kinase to convert NDPs to NTPs. See,e.g., Tables 5 and 6 for exemplary enzymes.

In some embodiments, the polyphosphate kinase belongs to thepolyphosphate kinase 2 (PPK2) family. In some embodiments, thepolyphosphate kinase belongs to a Class I PPK2 family, which transfershigh-energy phosphate from polyphosphate to NDPs to form NTPs. ATPproduced by the system is used as a high-energy phosphate donor toconvert NMPs to NDPs. In some embodiments, the polyphosphate kinasebelongs to a Class III PPK2 family, which transfers high-energyphosphate from polyphosphate to NMPs and NDPs to form NTPs. In someembodiments, Class III PPK2 is used alone to produce NTPs from NMPs. Inother embodiments, Class III PPK2 is used in combination with otherkinases. Class III PPK2 produces ATP from ADP, AMP, and polyphosphate,which is subsequently used by NMP and NDP kinases to convert NMPs toNTPs.

Non-limiting examples of PPK2 enzymes for use as provided herein arelisted in Table 6 (SEQ ID NO: 8-18). Thus, in some embodiments, the PPK2enzymes are thermostable. For example, the PPK2 enzymes may bethermostable Class III PPK2 enzymes, which favor ATP synthesis overpolyphosphate polymerization, and convert both ADP and AMP to ATP. Insome embodiments, the PPK2 enzymes are used to convert a polyphosphate,such as hexametaphosphate to ATP, at rates ranging, for example, from 10to 800 mM per hour (e.g., 10, 15, 20, 25, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 mM per hour).

In some embodiments, the RNA biosynthesis methods of the presentdisclosure utilize a PPK2 enzyme that comprises an amino acid sequenceidentical to the amino acid sequence identified by any one of SEQ ID NO:8-18. In some embodiments, the PPK2 enzyme comprises an amino acidsequence that is at least 70% identical to the amino acid sequenceidentified by any one of SEQ ID NO: 8-18. For example, the PPK2 enzymemay comprise an amino acid sequence that is at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the amino acid sequenceidentified by any one of SEQ ID NO: 8-18.

The present disclosure also encompasses fusion enzymes. Fusion enzymesmay exhibit multiple activities, each corresponding to the activity of adifferent enzyme. For example, rather than using an independentnucleoside monophosphate kinase and an independent nucleosidediphosphate kinase, a fusion enzyme (or any other enzyme) having bothnucleoside monophosphate kinase activity and nucleoside diphosphatekinase activity may be used.

TABLE 5 Examples of Pathway Enzymes Sequence Identification Enz. #Enzyme Name EC # Organism Uniprot # Number 1 RNase R 3.1.13.-Escherichia coli P21499 SEQ ID NO: 1 2 PPK1 2.7.4.1 ThermosynechococcusQ8DMA8 SEQ ID NO: 2 elongatus 3 UMP Kinase 2.7.4.22 Pyrococcus furiosusQ8U122 SEQ ID NO: 3 4 CMP Kinase 2.7.4.25 Thermus thermophilus Q5SL35SEQ ID NO: 4 5 GMP Kinase 2.7.4.8 Thermatoga maritima Q9X215 SEQ ID NO:5 6 AMP Kinase 2.7.4.3 Thermus thermophilus Q72I25 SEQ ID NO: 6 7 NDPKinase 2.7.4.6 Aquifex aeolicus O67528 SEQ ID NO: 7 8 RNA Polymerase

TABLE 6 Examples of PPK2 Enzymes Sequence Identification OrganismAccession # Number Meiothermus ruber DSM 1279 ADD29239.1 SEQ ID NO: 8Meiothermus silvanus DSM 9946 WP_013159015.1 SEQ ID NO: 9 Deinococcusgeothermalis DSM 11300 WP_011531362.1 SEQ ID NO: 10 Thermosynechococcuselongatus BP-1 NP_682498.1 SEQ ID NO: 11 Anaerolinea thermophila UNI-1WP_013558940 SEQ ID NO: 12 Caldilinea aerophila DSM 14535 WP_014433181SEQ ID NO: 13 Chlorobaculum tepidum TLS NP_661973.1 SEQ ID NO: 14Oceanithermus profundus DSM 14977 WP_013458618 SEQ ID NO: 15 Roseiflexuscastenholzii DSM 13941 WP_012120763 SEQ ID NO: 16 Roseiflexus sp. RS-1WP_011956376 SEQ ID NO: 17 Truepera radiovictrix DSM 17093 WP_013178933SEQ ID NO: 18

Polymerization of Nucleoside Triphosphates to Ribonucleic Acid

The final step in the biosynthesis of a RNA of interest is thepolymerization of NTPs to the RNA (e.g., dsRNA or ssRNA) end productusing, for example, a DNA-dependent RNA polymerase. In this step of theprocess, a DNA designed to encode the RNA of interest serves as thetemplate for the synthesis of the RNA of interest. The DNA template maybe engineered, in some instances, to have a transcriptional promoterthat selectively drives transcription of the RNA of interest. An exampleDNA template is shown in FIG. 3A. The DNA template encodes three RNAdomains: a sense domain (domain 1), a flexible hinge domain (domain 2)and a domain complementary to the sense domain (antisense domain 3).Following transcription of the DNA template, the antisense domain binds(hybridizes) to the sense domain to form a double-stranded RNA hairpinstem domain and an adjacent hairpin loop domain. Other examples of a DNAtemplate are shown in FIGS. 3B-3E. The DNA template in FIG. 3B containsconverging promoter sequences on complementary strands. RNA sequencestranscribed from each template strand anneal after transcription. TheDNA template in FIG. 3C, encoded as part of a plasmid, containsconverging promoter sequences on complementary strands, as well as oneor more terminator sequences to minimize read-through transcription. TheDNA template in FIG. 3D, encoded as part of a plasmid, containsindependent promoter-terminator cassettes driving transcription ofcomplementary sequences, which anneal after transcription. The DNAtemplate in FIG. 3E encodes a single RNA domain. Use of bothDNA-dependent RNA polymerase and RNA-dependent RNA polymerase produces adouble-stranded RNA end product.

Polymerization of RNA requires NTPs, a DNA template comprising atranscriptional promoter, and a polymerase (RNA polymerase) specific tothe transcriptional promoter. Typically, a polymerase for use asprovided herein is a single subunit polymerase, is highly selective forits cognate transcriptional promoters, has high fidelity, and is highlyefficient. Examples of polymerases include, without limitation, T7 RNApolymerase, T3 RNA polymerase, and SP6 RNA polymerase. Bacteriophage T7RNA polymerase is a DNA-dependent RNA polymerase that is highly specificfor the T7 phage promoters. The 99 KD enzyme catalyzes in vitro RNAsynthesis from a cloned DNA sequence under control of the T7 promoter.Bacteriophage T3 RNA polymerase is a DNA-dependent RNA polymerase thatis highly specific for the T3 phage promoters. The 99 KD enzymecatalyzes in vitro RNA synthesis from a cloned DNA sequence under the T3promoter. Bacteriophage SP6 RNA polymerase is a DNA-dependent RNApolymerase that is highly specific for the SP6 phage promoter. The 98.5KD polymerase catalyzes in vitro RNA synthesis from a cloned DNAtemplate under the SP6 promoter. Each of T7, T3, and SP6 polymerase areoptimally active at 37-40° C. In some embodiments, thermostable variantsof T7, T3, and SP6 polymerase are used. Thermostable variant polymerasesare typically optimally active at temperatures above 40° C. (or about50-60° C.).

“Conditions that result in production of nucleoside triphosphates andpolymerization of the nucleoside triphosphates,” also referred to as“conditions for the biosynthesis of RNA,” may be determined by one ofordinary skill in the art, taking into consideration, for example,optimal conditions for polymerase activity, including pH, temperature,length of time, and salt concentration of the cell lysate as well as anyexogenous cofactors.

The pH of a cell lysate during the biosynthesis of RNA may have a valueof 3.0 to 8.0. In some embodiments, the pH value of a cell lysate is3.0-8.0, 4.0-8.0, 5.0-8.0, 6.0-8.0, 7.0-8.0, 3.0-7.0, 4.0-7.0, 5.0-7.0,6.0-7.0, 3.0-6.0, 4.0-6.0, 5.0-6.0, 3.0-5.0, 3.0-4.0, or 4.0-5.0. Insome embodiments, the pH value of a cell lysate is 3.0, 3.5, 4.0, 4.5,5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, the pH valueof a cell lysate during biosynthesis of RNA is 7.0.

The temperature of a cell lysate during biosynthesis of RNA may be 15°C. to 70° C. In some embodiments, the temperature of a cell lysateduring biosynthesis of RNA is 15-60° C., 15-50° C., 15-40° C., 15-30°C., 25-70° C., 25-60° C., 25-50° C., 25-40° C., 30-70° C., 30-60° C.,30-50° C., 40-70° C., 40-60° C., 40-50° C., 50-70° C., or 50-60° C. Insome embodiments, the temperature of a cell lysate during biosynthesisof RNA is 15° C., 25° C., 32° C., 37° C., 42° C., 45° C., 55° C., 56°C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65°C., 66° C., 67° C., 68° C., 69° C., or 70° C. In some embodiments, thetemperature of a cell lysate during biosynthesis of RNA is 50° C.

A cell lysate during biosynthesis of RNA may be incubated for 15 minutes(min) to 72 hours (hrs). In some embodiments, a cell lysate duringbiosynthesis of RNA is incubated for 30 min-48 hrs. For example, a celllysate during biosynthesis of RNA may be incubated for 30 min, 45 min, 1hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11hrs, 12 hrs, 18 hrs, 24 hrs, 30 hrs, 36 hrs, 42 hours, or 48 hours. Insome embodiments, a cell lysate during biosynthesis of RNA is incubatedfor 3 hours. In some embodiments, a cell lysate during biosynthesis ofRNA is incubated for 24 hours at a temperature of 37° C.

In some embodiments, a cell lysate during biosynthesis of RNA isincubated at a pH of 7.0 for 2-4 hours at a temperature of 50° C.

Some polymerase activities may require the presence of metal ions. Thus,in some embodiments, metal ions are added to a cell lysate. Non-limitingexamples of metal ions include Mg²⁺, Li⁺, Na⁺, K⁺, Ni²⁺, Ca²⁺, Cu²⁺, andMn²⁺. Other metal ions may be used. In some embodiments, more than onemetal ion may be used. The concentration of a metal ion in a cell lysatemay be 0.1 mM to 100 mM, or 10 mM to 50 mM. In some embodiments, theconcentration of a metal ion in a cell lysate is 0.1, 0.2, 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5., 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0,70.0, 80.0, 90.0, or 100.0 mM.

In some embodiments, salt is added to a cell lysate, for example, toprevent enzyme aggregation. For example, sodium chloride, potassiumchloride, sodium acetate, potassium acetate, or a combination thereof,may be added to a cell lysate. The concentration of salt in a celllysate during a RNA depolymerization reaction may be 5 mM to 1 M. Insome embodiments, the concentration of salt in a cell lysate during aRNA depolymerization reaction 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM,100 mM, 150 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M.

Thermostable Enzymes

One advantage of the cell-free RNA-biosynthesis methods of the presentdisclosure is that all of the enzymes needed to convert endogenous RNAto synthetic double-stranded RNA, for example, may be (but need not be)expressed in a single engineered cell. For example, a clonal populationof the engineered cell is cultured to a desired cell density, the cellsare lysed, incubated under conditions that result in depolymerization ofendogenous RNA to its monomer form (e.g., at a temperature of 30-37°C.), subjected to temperatures sufficient to inactivate endogenousnucleases and phosphatases (e.g., 40-90° C.), and incubated underconditions that result in the polymerization of RNA (e.g., dsRNA orssRNA) (e.g., 30-50° C.). In order to proceed to end product syntheticRNA, the enzymes required for conversion of NMPs to NDPs (e.g.,nucleoside monophosphate kinases and/or polyphosphate kinases), fromNDPs to NTPs (e.g., nucleoside diphosphate kinases and/or polyphosphatekinase), and from NTPs to RNA (e.g., polymerase) should be thermostableto avoid denaturation during heat inactivation of the endogenousnuclease (and/or exogenous nucleases) and phosphatases. Thermostabilityrefers to the quality of enzymes to resist denaturation at relativelyhigh temperature. For example, if an enzyme is denatured (inactivated)at a temperature of 42° C., an enzyme having similar activity (e.g.,kinase activity) is considered “thermostable” if it does not denature at42° C.

An enzyme (e.g., kinase or polymerase) is considered thermostable if theenzyme (a) retains activity after temporary exposure to hightemperatures that denature other native enzymes or (b) functions at ahigh rate after temporary exposure to a medium to high temperature wherenative enzymes function at low rates.

In some embodiments, a thermostable enzyme retains greater than 50%activity following temporary exposure to relatively high temperature(e.g., higher than 41° C. for kinases obtained from E. coli, higher than37° C. for many RNA polymerases) that would otherwise denature a similar(non-thermostable) native enzyme. In some embodiments, a thermostableenzyme retains 50-100% activity following temporary exposure torelatively high temperature that would otherwise denature a similar(non-thermostable) native enzyme. For example, a thermostable enzyme mayretain 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, or 50-55%activity following temporary exposure to relatively high temperaturethat would otherwise denature a similar (non-thermostable) nativeenzyme. In some embodiments, a thermostable enzyme retains 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, or 100% activity following temporary exposure to relatively hightemperature that would otherwise denature a similar (non-thermostable)native enzyme.

In some embodiments, the activity of a thermostable enzyme aftertemporary exposure to medium to high temperature (e.g., 42-80° C.) isgreater than (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% greater than) the activity ofa similar (non-thermostable) native enzyme.

The activity of a thermostable kinase, for example, may be measured bythe amount of NMP or NDP the kinase is able to phosphorylate. Thus, insome embodiments, a thermostable kinase, at relatively high temperature(e.g., 42° C.) converts greater than 50% of NMP to NDP, or greater than50% of NDP to NTP, in the same amount of time required to complete asimilar conversion at 37° C. In some embodiments, a thermostable kinase,at relatively high temperature (e.g., 42° C.) converts greater than 60%of NMP to NDP, or greater than 60% of NDP to NTP, in the same amount oftime required to complete a similar conversion at 37° C. In someembodiments, a thermostable kinase, at relatively high temperature(e.g., 42° C.) converts greater than 70% of NMP to NDP, or greater than70% of NDP to NTP, in the same amount of time required to complete asimilar conversion at 37° C. In some embodiments, a thermostable kinase,at relatively high temperature (e.g., 42° C.) converts greater than 80%of NMP to NDP, or greater than 80% of NDP to NTP, in the same amount oftime required to complete a similar conversion at 37° C. In someembodiments, a thermostable kinase, at relatively high temperature(e.g., 42° C.) converts greater than 90% of NMP to NDP, or greater than90% of NDP to NTP, in the same amount of time required to complete asimilar conversion at 37° C.

The activity of a thermostable polymerase, for example, is assessedbased on fidelity and polymerization kinetics (e.g., rate ofpolymerization). Thus, one unit of a thermostable T7 polymerase, forexample, may incorporate 10 nmoles of NTP into acid insoluble materialin 30 minutes at temperatures above 37° C. (e.g., at 50° C.).

Thermostable enzymes (e.g., kinases or polymerases) may remain active(able to catalyze a reaction) at a temperature of 42° C. to 80° C., orhigher. In some embodiments, thermostable enzymes remain active at atemperature of 42-80° C., 42-70° C., 42-60° C., 42-50° C., 50-80° C.,50-70° C., 50-60° C., 60-80° C., 60-70° C., or 70-80° C. For example,thermostable enzymes may remain active at a temperature of 42° C., 43°C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52°C., 53° C., 54° C., 55° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60°C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69°C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78°C., 79° C., or 80° C. Thermostable enzymes may remain active atrelatively high temperatures for 15 minutes to 48 hours, or longer. Forexample, thermostable enzymes may remain active at relatively hightemperatures for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 24, 36, 42, or 48 hours.

Non-limiting examples of thermostable NMP kinases are listed in Tables 5and 7. Other thermostable kinases include thermostable nucleosidediphosphate kinases, thermostable pyruvate kinases, and thermostablepolyphosphate kinases (see, e.g., Table 6). Other thermostable kinasesare encompassed by the present disclosure.

TABLE 7 Examples of Thermostable Nucleoside Monophosphate Kinases.Enzyme Name Host Organism EC # Reaction Uniprot Ref Uridylate Tthermophilus, 2.7.4.22 UMP → UDP + P43891 41 kinase P. furiosus ADPQ8U122 42, 43 Cytidylate T thermophilus, 2.7.4.25 CMP → CDP + Q5SL35 41kinase P. furiosus ADP Q8U2L4 46 Guanylate T thermophilus, 2.7.4.8 GMP →GDP + Q5SI18 41 kinase T. maritima ADP Q9X215 49 Adenylate Tthermophilus, 2.7.4.3 AMP → 2ADP Q72125 52, kinase P. furiosus 53 Q8U20746

Non-limiting examples of RNA polymerases are listed in Table 8. OtherRNA polymerases, including thermostable RNA polymerases, are encompassedby the present disclosure.

TABLE 8 Examples of RNA Polymerases Host Enzyme Name Organism FunctionUniprot Ref T7 RNA T7 Phage DNA-dependent P00573 54, Polymerase RNApolymerase 55 Φ6 RdRP Phage Φ6 RNA-dependent P11124 56 RNA polymerase T3RNA T3 Phage DNA-dependent P07659 57 polymerase RNA polymerase SP6Polymerase SP6 Phage DNA-Dependent P06221 58 RNA polymerase

Thermostable RNA polymerases may be prepared by modifying wild-typeenzymes. Such modifications (e.g., mutations) are known. For example,variant thermostable T7 RNA polymerases may include one or more of thefollowing point mutations: V426L, A702V, V795I, S430P, F849I, S633I,F880Y, C510R, and S767G (EP2377928 and EP1261696A1, each of which isincorporated herein by reference). In some embodiments, a variantthermostable T7 RNA polymerase includes V426L, A702V, and V795Imutations. In some embodiments, a variant thermostable T7 RNA polymeraseincludes S430P, F849I, S633I, and F880Y mutations. In some embodiments,a variant thermostable T7 RNA polymerase includes F880Y, S430P, F849I,S633I, C510R, and S767G mutations. In some embodiments, a variantthermostable T7 RNA polymerase includes Y639V, H784G, E593G, and V685Amutations. In some embodiments, a variant thermostable T7 RNA polymeraseincludes S430P, N433T, S633P, F849I, and F880Y mutations. Other variantand recombinant thermostable polymerases are encompassed by the presentdisclosure.

In some embodiments, a thermostable T7 polymerase is used to produce aRNA of interest. For example, a thermostable T7 polymerase (e.g.,incubated at a temperature of 40-60° C.) having a concentration of 1-2%total protein may be used to synthesize RNA of interest at a rate ofgreater than 2 g/L/hr (or, e.g., 2 g/L/hr-10 g/L/hr). As anotherexample, a thermostable T7 polymerase (e.g., incubated at a temperatureof 40-60° C.) having a concentration of 3-5% total protein may be usedto synthesize RNA of interest at a rate of greater than 10 g/L/hr (or,e.g., 10 g/L/hr-20 g/L/hr).

It should be understood that while many embodiments of the presentdisclosure describe the use of thermostable polymerases/enzymes, otherenzymes/polymerases may be used. In some embodiments, polymerase may beexogenously added to heat-inactivated cell lysates, for example, tocompensate for any reduction or loss of activity of the thermostableenzyme(s).

RNA of Interest

Methods of the present disclosure are used to biosynthesize a RNA ofinterest. The RNA may be single-stranded or double-stranded. In someembodiments, the RNA is a double-stranded RNA interference molecule. Forexample, a RNA of interest may be an siRNA or a hairpin RNA interferencemolecule. As discussed above, a RNA of interest is encoded by a DNAtemplate, examples of which are shown in FIGS. 3A-3E. The RNA producedusing the template of FIG. 3A includes a sense domain (domain 1), aflexible hinge domain (domain 2) and a domain complementary to the sensedomain (antisense domain 3). Following transcription of the DNAtemplate, the antisense domain binds (hybridizes) to the sense domain toform a double-stranded RNA hairpin stem domain and an adjacent hairpinloop (hinge) domain.

A double-stranded hairpin stem domain is formed by the binding of twocomplementary nucleic acid domains (e.g., discrete nucleotide sequences)to each other. Nucleic acid domains are “complementary” if they bind(base pair via Watson-Crick interactions, hybridize) to each other toform a double-stranded nucleic acid. The complementary domains of a DNAtemplate encoding a RNA of interest may vary, depending, for example, onthe desired end product. Complementary domains may have a length of, forexample, 4 to 1000 nucleotides, or longer. For example, complementarydomains may have a length of 4 to 10, 4 to 20, 4 to 30, 4 to 50, 4 to60, 4 to 70, 4 to 80, 4 to 90, 4 to 100, 4 to 200, 4 to 300, 4 to 400,or 4 to 500, or 4 to 1000 nucleotides. In some embodiments complementarydomains have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides. In some embodiments, complementary domains have a length of4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 nucleotides.

A hairpin loop domain is also formed by binding of two complementarynucleic acid domains. A hairpin loop domain is the intervening sequencebetween two complementary domains. Typically, a hairpin loop domain isnon-specific, meaning that it is not designed to bind intramolecularlyor to another nucleic acid. A hairpin loop domain forms a loop-likestructure upon binding of the complementary domains to form adouble-stranded hairpin stem domain. In some embodiments, a hairpin loopdomain has a length of 4 to 500 nucleotides, or more. For example, ahairpin loop domain may have a length of 4 to 10, 4 to 20, 4 to 30, 4 to50, 4 to 60, 4 to 70, 4 to 80, 4 to 90, 4 to 100, 4 to 200, 4 to 300, 4to 400, or 4 to 500 nucleotides. In some embodiments, a hairpin loopdomain has a length of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides.

A “double-stranded RNA” of the present disclosure encompasses whollydouble-stranded molecules, which do not contain a single-stranded region(e.g., a loop or overhang), as well as partially double-strandedmolecules, which contain a double-stranded region and a single-strandedregion (e.g., a loop or overhang). The dsRNA product depicted at thebottom of FIG. 3A is considered a partially double-stranded molecule,while the dsRNA product depicted at the bottom of FIG. 3B is considereda wholly double-stranded molecule.

Examples of “single-stranded RNA” of interest include messenger RNA(mRNA) and antisense RNA. Thus, provided herein are methods ofsynthesizing mRNA and other single-stranded RNA molecules.

These methods may comprise (a) lysing cultured engineered cells thatcomprise RNA, an enzyme that depolymerizes RNA, thermostable kinases, athermostable RNA polymerase, thereby producing a cell lysate, (b)incubating the cell lysate produced in step (a) under conditions thatresult in depolymerization of RNA, thereby producing a cell lysate thatcomprises nucleoside monophosphates, (c) heating the cell lysateproduced in step (b) to a temperature that inactivates endogenousnucleases and phosphatases without inactivating the thermostable kinasesand thermostable RNA polymerase, thereby producing a cell lysate thatcomprises heat-inactivated nucleases and phosphatases, and (d)incubating the cell lysate produced in (c) in the presence of an energysource and an engineered DNA template containing a promoter operablylinked to a nucleotide sequence encoding a RNA of interest, underconditions that result in production of nucleoside triphosphates andpolymerization of the nucleoside triphosphates, thereby producing a celllysate that comprises the mRNA of interest.

Alternatively, such methods may comprise (a) combining cell lysatesobtained from engineered cells that comprise endogenous, polymeric RNA,an enzyme that depolymerizes RNA, thermostable nucleoside monophosphate(NMP) kinases, thermostable nucleoside diphosphate (NDP) kinases, athermostable PPK2 kinase, and/or a polyphosphate, to produce a celllysate mixture, (b) incubating the cell lysate mixture produced in step(a) under conditions that result in depolymerization of RNA, therebyproducing a cell lysate that comprises nucleoside monophosphates, (c)heating the cell lysate produced in step (b) to a temperature thatinactivates phosphatases and RNases (and any other activities that maybe detrimental to RNA stability or polymerization fidelity, such asnative RNA polymerase, NMP reductases, and/or nucleosidases) withoutinactivating the thermostable kinase and thermostable RNA polymerase,thereby producing a cell lysate that comprises heat-inactivatedphosphatases and RNases (and other deleterious cellular activities), and(d) incubating the cell lysate produced in step (c) in the presence ofan energy source and an engineered DNA template containing a promoteroperably linked to a nucleotide sequence encoding a RNA of interest,under conditions that result in production of nucleoside triphosphatesand polymerization of the nucleoside triphosphates, thereby producing acell lysate that comprises mRNA.

In some embodiments, the DNA template encoding the RNA containing asingle target domain is transcribed using a DNA-dependent RNApolymerase, such as, for example, a T7 RNA polymerase, and the resultingRNA transcript serves as a template for a RNA-dependent RNA polymerase,such as, for example, the phage ϕ6 RdRP, to synthesize a complementaryRNA molecule, yielding a dsRNA. See, e.g., FIG. 3B. Phage ϕ6 is a doublestranded RNA virus that infects members of the genus Pseudomonas. Thisphage encodes an RdRP that is capable of synthesizing RNA using a RNAtemplate, yielding a dsRNA molecule. The ϕ6 RdRP is capable ofpolymerizing RNA absent a primer molecule, thus the polymerase requiresonly template RNA (Wright, S. et al, 2012. Journal of Virology. March;86(5):2837-49; Van Dijk, A A., et al, 2004. J Gen Virol. May; 85(Pt 5),incorporated herein by reference). Other RNA-dependent RNA polymerase(RdRP) are encompassed by the present disclosure.

In some embodiments, the engineered cells comprise a DNA templateencoding the RNA of interest. A DNA template encoding the RNA may beintegrated into the genomic DNA of the engineered cells, or a DNAtemplate may be introduced into the engineered cells on a plasmid. Inother embodiments, the DNA template is added to the cell lysate duringbiosynthesis of the RNA of interest (e.g., following a heat inactivationstep). In some embodiments, the concentration of the DNA template in acell lysate is 0.05-1 μg/μl. In some embodiments, the concentration ofthe DNA template in a cell lysate is 0.05 μg/μl, 0.1 μg/μl, 0.5 μg/μl,1.0 μg/μl.

As discussed above, other examples of RNA end products of interestinclude messenger RNA (mRNA) and short/small-interfering RNA (siRNA) (asynthetic RNA duplex designed to specifically target a particular mRNAfor degradation).

In some embodiments, the concentration of RNA end product(biosynthesized RNA of interest) is at least 1 g/L to 50 g/L of celllysate. For example, the concentration of RNA end product may be 1, 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 g/L, or more.

In some embodiments, a RNA of interest is designed to bind to a targetnucleic acid of interest and is used, for example, as a therapeutic,prophylactic, or diagnostic agent.

Protease Targeting

Engineered cells of the present disclosure may express (e.g.,endogenously express) enzymes necessary for the health of the cells thatmay have a negative impact on the production of nucleic acids, such asRNA. Such enzymes are referred to herein as “target enzymes.” Forexample, target enzymes expressed by engineered cells may compete forsubstrates or cofactors with an enzyme that increases the rate ofprecursor supplied to a RNA biosynthetic pathway. As another example,target enzymes expressed by the engineered cells may compete forsubstrates or cofactors with an enzyme that is a key pathway entryenzyme of a RNA biosynthetic pathway. As yet another example, targetenzymes expressed by the engineered cells may compete for substrates orcofactors with an enzyme that supplies a substrate or cofactor of a RNAbiosynthetic pathway.

To negate, or reduce, this negative impact, target enzymes can bemodified to include a site-specific protease-recognition sequence intheir protein sequence such that the target enzyme may be “targeted” andcleaved for inactivation during RNA production (see, e.g., U.S.Publication No. 2012/0052547 A1, published on Mar. 1, 2012; andInternational Publication No. WO 2015/021058 A2, published Feb. 12,2015, each of which is incorporated by reference herein).

Cleavage of a target enzyme containing a site-specificprotease-recognition sequence results from contact with a cognatesite-specific protease is sequestered in the periplasm of cell (separatefrom the target enzyme) during the cell growth phase (e.g., asengineered cells are cultured) and is brought into contact with thetarget enzyme during the RNA production phase (e.g., following celllysis to produce a cell lysate). Thus, engineered cells of the presentdisclosure comprise, in some embodiments, (i) an engineered nucleic acidencoding a target enzyme that negatively impacts the rate of RNAproduction and includes a site-specific protease-recognition sequence inthe protein sequence of the target enzyme, and (ii) an engineerednucleic acid encoding a site-specific protease that cleaves thesite-specific protease-recognition sequence of the target enzyme andincludes a periplasmic-targeting sequence. This periplasmic-targetingsequence is responsible for sequestering the site-specific protease tothe periplasmic space of the cell until the cell is lysed. Examples ofperiplasmic-targeting sequences are provided below.

Examples of proteases that may be used in accordance with the presentdisclosure include, without limitation, alanine carboxypeptidase,proteases obtained from Armillaria mellea, astacin, bacterial leucylaminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosolalanyl aminopeptidase, elastase, endoproteinase Brg-C, enterokinase,gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase,human rhinovirus 3C protease, hypodermin C, Iga-specific serineendopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC,lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionylaminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E,picornain 2B, picornain 3C, proendopeptidase, prolyl aminopeptidase,proprotein convertase I, proprotein convertase II, russellysin,saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissuekallikrein, proteases obtained from tobacco etch virus (TEV), togavirin,tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin B,venombin BB and Xaa-pro aminopeptidase.

Periplasmic Targeting

Enzymes of a nucleic acid (e.g., RNA) biosynthetic pathway may includeat least one enzyme that has a negative impact on the health (e.g.,viability) of a cell. To negate or reduce this negative impact, anenzyme can be modified to include a relocation sequence such that theenzyme is relocated to a cellular or extra-cellular compartment where itis not naturally located and where the enzyme does not negatively impactthe health of the cell (see, e.g., Publication No. US-2011-0275116-A1,published on Nov. 10, 2011, incorporated by reference herein). Forexample, an enzyme of a biosynthetic pathway may be relocated to theperiplasmic space of a cell.

Thus, in some embodiments, engineered cells of the present disclosurecomprise at least one enzyme of a nucleic acid (e.g., RNA) biosyntheticpathway that is linked to a periplasmic-targeting sequence. A“periplasmic-targeting sequence” is an amino acid sequence that targetsto the periplasm of a cell the protein to which it is linked. A proteinthat is linked to a periplasmic-targeting sequence will be sequesteredin the periplasm of the cell in which the protein is expressed.

Periplasmic-targeting sequences may be derived from the N-terminus ofbacterial secretory protein, for example. The sequences vary in lengthfrom about 15 to about 70 amino acids. The primary amino acid sequencesof periplasmic-targeting sequences vary, but generally have a commonstructure, including the following components: (i) the N-terminal parthas a variable length and generally carries a net positive charge; (ii)following is a central hydrophobic core of about 6 to about 15 aminoacids; and (iii) the final component includes four to six amino acidswhich define the cleavage site for signal peptidases.

Periplasmic-targeting sequences of the present disclosure, in someembodiments, may be derived from a protein that is secreted in a Gramnegative bacterium. The secreted protein may be encoded by thebacterium, or by a bacteriophage that infects the bacterium. Examples ofGram negative bacterial sources of secreted proteins include, withoutlimitation, members of the genera Escherichia, Pseudomonas, Klebsiella,Salmonella, Caulobacter, Methylomonas, Acetobacter, Achromobacter,Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Azotobacter,Burkholderia, Citrobacter, Comamonas, Enterobacter, Erwinia, Rhizobium,Vibrio, and Xanthomonas.

Examples of periplasmic-targeting sequences for use in accordance withthe present disclosure include, without limitation, sequences selectedfrom the group consisting of: MKIKTGARILALSALTTMMFSASALA (SEQ ID NO:19); MKQSTIALALLPLLFTPVTKA (SEQ ID NO: 20); MMITLRKLPLAVAVAAGVMSAQAMA(SEQ ID NO: 21); MNKKVLTLSAVMASMLFGAAAHA (SEQ ID NO: 22);MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 23); MKKIWLALAGLVLAFSASA (SEQ ID NO:24); MMTKIKLLMLIIFYLIISASAHA (SEQ ID NO: 25); MKQALRVAFGFLILWASVLHA (SEQID NO: 26); MRVLLFLLLSLFMLPAFS (SEQ ID NO: 27); andMANNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 28).

Engineered Cells

Engineered cells of the present disclosure typically comprise at leastone, most, or all, of the enzymatic activities required to biosynthesizeRNA. “Engineered cells” are cells that comprise at least one engineered(e.g., recombinant or synthetic) nucleic acid, or are otherwise modifiedsuch that they are structurally and/or functionally distinct from theirnaturally-occurring counterparts. Thus, a cell that contains anengineered nucleic acid is considered an “engineered cell.”

Engineered cells of the present disclosure, in some embodiments,comprise RNA, enzymes that depolymerizes RNA, thermostable kinases,and/or thermostable polymerases. In some embodiments, the engineeredcells further comprise a DNA template containing a promoter operablylinked to a nucleotide sequence encoding a RNA of interest.

Engineered cells, in some embodiments, express selectable markers.Selectable markers are typically used to select engineered cells thathave taken up and expressed an engineered nucleic acid followingtransfection of the cell (or following other procedure used to introduceforeign nucleic acid into the cell). Thus, a nucleic acid encodingproduct may also encode a selectable marker. Examples of selectablemarkers include, without limitation, genes encoding proteins thatincrease or decrease either resistance or sensitivity to antibiotics(e.g., ampicillin resistance genes, kanamycin resistance genes, neomycinresistance genes, tetracycline resistance genes and chloramphenicolresistance genes) or other compounds. Additional examples of selectablemarkers include, without limitation, genes encoding proteins that enablethe cell to grow in media deficient in an otherwise essential nutrient(auxotrophic markers). Other selectable markers may be used inaccordance with the present disclosure.

An engineered cell “expresses” a product if the product, encoded by anucleic acid (e.g., an engineered nucleic acid), is produced in thecell. It is known in the art that gene expression refers to the processby which genetic instructions in the form of a nucleic acid are used tosynthesize a product, such as a protein (e.g., an enzyme).

Engineered cells may be prokaryotic cells or eukaryotic cells. In someembodiments, engineered cells are bacterial cells, yeast cells, insectcells, mammalian cells, or other types of cells.

Engineered bacterial cells of the present disclosure include, withoutlimitation, engineered Escherichia spp., Streptomyces spp., Zymomonasspp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobiumspp., Clostridium spp., Corynebacterium spp., Streptococcus spp.,Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp.,Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp.,Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacterspp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp.,Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacteriumspp., Serratia spp., Saccharopolyspora spp., Thermus spp.,Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp.,Saccharopolyspora spp., Agrobacterium spp., and Pantoea spp.

Engineered yeast cells of the present disclosure include, withoutlimitation, engineered Saccharomyces spp., Schizosaccharomyces,Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

In some embodiments, engineered cells of the present disclosure areengineered Escherichia coli cells, Bacillus subtilis cells, Pseudomonasputida cells, Saccharomyces cerevisae cells, or Lactobacillus breviscells. In some embodiments, engineered cells of the present disclosureare engineered Escherichia coli cells.

Engineered Nucleic Acids

A “nucleic acid” is at least two nucleotides covalently linked together,and in some instances, may contain phosphodiester bonds (e.g., aphosphodiester “backbone”). Nucleic acids (e.g., components, orportions, of nucleic acids) may be naturally occurring or engineered.“Naturally occurring” nucleic acids are present in a cell that exists innature in the absence of human intervention. “Engineered nucleic acids”include recombinant nucleic acids and synthetic nucleic acids. A“recombinant nucleic acid” refers to a molecule that is constructed byjoining nucleic acid molecules (e.g., from the same species or fromdifferent species) and, typically, can replicate in a living cell. A“synthetic nucleic acid” refers to a molecule that is biologicallysynthesized, chemically synthesized, or by other means synthesized oramplified. A synthetic nucleic acid includes nucleic acids that arechemically modified or otherwise modified but can base pair withnaturally-occurring nucleic acid molecules. Recombinant and syntheticnucleic acids also include those molecules that result from thereplication of either of the foregoing. Engineered nucleic acids maycontain portions of nucleic acids that are naturally occurring, but as awhole, engineered nucleic acids do not occur naturally and require humanintervention. In some embodiments, a nucleic acid encoding a product ofthe present disclosure is a recombinant nucleic acid or a syntheticnucleic acid. In other embodiments, a nucleic acid encoding a product isnaturally occurring.

An engineered nucleic acid encoding RNA, as provided herein, may beoperably linked to a “promoter,” which is a control region of a nucleicacid at which initiation and rate of transcription of the remainder of anucleic acid are controlled. A promoter drives expression or drivestranscription of the nucleic acid that it regulates.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment of a given gene or sequence. Such apromoter can be referred to as “endogenous.”

In some embodiments, a coding nucleic acid sequence may be positionedunder the control of a recombinant or heterologous promoter, whichrefers to a promoter that is not normally associated with the encodedsequence in its natural environment. Such promoters may includepromoters of other genes; promoters isolated from any other cell; andsynthetic promoters or enhancers that are not “naturally occurring” suchas, for example, those that contain different elements of differenttranscriptional regulatory regions and/or mutations that alterexpression through methods of genetic engineering that are known in theart. In addition to producing nucleic acid sequences of promoters andenhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, includingpolymerase chain reaction (PCR).

A promoter is considered to be “operably linked” when it is in a correctfunctional location and orientation in relation to the nucleic acid itregulates to control (“drive”) transcriptional initiation and/orexpression of that nucleic acid.

Engineered nucleic acids of the present disclosure may contain aconstitutive promoter or an inducible promoter. A “constitutivepromoter” refers to a promoter that is constantly active in a cell. An“inducible promoter” refers to a promoter that initiates or enhancestranscriptional activity when in the presence of, influenced by, orcontacted by an inducer or inducing agent, or activated in the absenceof a factor that causes repression. Inducible promoters for use inaccordance with the present disclosure include any inducible promoterdescribed herein or known to one of ordinary skill in the art. Examplesof inducible promoters include, without limitation,chemically/biochemically-regulated and physically-regulated promoterssuch as alcohol-regulated promoters, tetracycline-regulated promoters,steroid-regulated promoters, metal-regulated promoters,pathogenesis-regulated promoters, temperature/heat-inducible,phosphate-regulated (e.g., PhoA), and light-regulated promoters.

An inducer or inducing agent may be endogenous or a normally exogenouscondition (e.g., light), compound (e.g., chemical or non-chemicalcompound) or protein that contacts an inducible promoter in such a wayas to be active in regulating transcriptional activity from theinducible promoter. Thus, a “signal that regulates transcription” of anucleic acid refers to an inducer signal that acts on an induciblepromoter. A signal that regulates transcription may activate orinactivate transcription, depending on the regulatory system used.Activation of transcription may involve directly acting on a promoter todrive transcription or indirectly acting on a promoter by inactivation arepressor that is preventing the promoter from driving transcription.Conversely, deactivation of transcription may involve directly acting ona promoter to prevent transcription or indirectly acting on a promoterby activating a repressor that then acts on the promoter.

Engineered nucleic acids may be introduced into host cells using anymeans known in the art, including, without limitation, transformation,transfection (e.g., chemical (e.g., calcium phosphate, cationicpolymers, or liposomes) or non-chemical (e.g., electroporation,sonoporation, impalefection, optical transfection, hydrodynamictransfection)), and transduction (e.g., viral transduction).

Enzymes or other proteins encoded by a naturally-occurring,intracellular nucleic acid may be referred to as “endogenous enzymes” or“endogenous proteins.”

Cell Cultures and Cell Lysates

Typically, engineered cells are cultured. “Culturing” refers to theprocess by which cells are grown under controlled conditions, typicallyoutside of their natural environment. For example, engineered cells,such as engineered bacterial cells, may be grown as a cell suspension inliquid nutrient broth, also referred to as liquid “culture medium.”

Examples of commonly used bacterial Escherichia coli growth mediainclude, without limitation, LB (Lysogeny Broth) Miller broth (1% NaCl):1% peptone, 0.5% yeast extract, and 1% NaCl; LB (Lysogeny Broth) LennoxBroth (0.5% NaCl): 1% peptone, 0.5% yeast extract, and 0.5% NaCl; SOBmedium (Super Optimal Broth): 2% peptone, 0.5% Yeast extract, 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄; SOC medium (Super Optimalbroth with Catabolic repressor): SOB+20 mM glucose; 2×YT broth (2× Yeastextract and Tryptone): 1.6% peptone, 1% yeast extract, and 0.5% NaCl; TB(Terrific Broth) medium: 1.2% peptone, 2.4% yeast extract, 72 mM K₂HPO₄,17 mM KH₂PO₄ and 0.4% glycerol; and SB (Super Broth) medium: 3.2%peptone, 2% yeast extract, and 0.5% NaCl and or Korz medium (Korz, D Jet al. 1995).

Examples of high density bacterial Escherichia coli growth mediainclude, but are not limited to, DNAGro™ medium, ProGro™ medium, AutoX™medium, DetoX™ medium, InduX™ medium, and SecPro™ medium.

In some embodiments, engineered cells are cultured under conditions thatresult in expression of enzymes or nucleic acids. Such cultureconditions may depend on the particular product being expressed and thedesired amount of the product.

In some embodiments, engineered cells are cultured at a temperature of30° C. to 40° C. For example, engineered cells may be cultured at atemperature of 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C.,37° C., 38° C., 39° C. or 40° C. Typically, engineered cells, such asengineered E. coli cells, are cultured at a temperature of 37° C.

In some embodiments, engineered cells are cultured for a period of timeof 12 hours to 72 hours, or more. For example, engineered cells may becultured for a period of time of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66,or 72 hours. Typically, engineered cells, such as engineered bacterialcells, are cultured for a period of time of 12 to 24 hours. In someembodiments, engineered cells are cultured for 12 to 24 hours at atemperature of 37° C.

In some embodiments, engineered cells are cultured (e.g., in liquid cellculture medium) to an optical density, measured at a wavelength of 600nm (OD600), of 5 to 200. In some embodiments, engineered cells arecultured to an OD600 of 5, 10, 15, 20, 25, 50, 75, 100, 150, or 200.

In some embodiments, engineered cells are cultured to a density of 1×10⁸(OD<1) to 2×10¹¹ (OD ˜200) viable cells/ml cell culture medium. In someembodiments, engineered cells are cultured to a density of 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹,4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹ 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, or 2×10¹¹ viablecells/ml. (Conversion factor: OD 1=8×10⁸ cells/ml).

In some embodiments, engineered cells are cultured in a bioreactor. Abioreactor refers simply to a container in which cells are cultured,such as a culture flask, a dish, or a bag that may be single-use(disposable), autoclavable, or sterilizable. The bioreactor may be madeof glass, or it may be polymer-based, or it may be made of othermaterials.

Examples of bioreactors include, without limitation, stirred tank (e.g.,well mixed) bioreactors and tubular (e.g., plug flow) bioreactors,airlift bioreactors, membrane stirred tanks, spin filter stirred tanks,vibromixers, fluidized bed reactors, and membrane bioreactors. The modeof operating the bioreactor may be a batch or continuous processes andwill depend on the engineered cells being cultured. A bioreactor iscontinuous when the feed and product streams are continuously being fedand withdrawn from the system. A batch bioreactor may have a continuousrecirculating flow, but no continuous feeding of nutrient or productharvest. For intermittent-harvest and fed-batch (or batch fed) cultures,cells are inoculated at a lower viable cell density in a medium that issimilar in composition to a batch medium. Cells are allowed to growexponentially with essentially no external manipulation until nutrientsare somewhat depleted and cells are approaching stationary growth phase.At this point, for an intermittent harvest batch-fed process, a portionof the cells and product may be harvested, and the removed culturemedium is replenished with fresh medium. This process may be repeatedseveral times. For production of recombinant proteins and antibodies, afed-batch process may be used. While cells are growing exponentially,but nutrients are becoming depleted, concentrated feed medium (e.g.,10-15 times concentrated basal medium) is added either continuously orintermittently to supply additional nutrients, allowing for furtherincrease in cell concentration and the length of the production phase.Fresh medium may be added proportionally to cell concentration withoutremoval of culture medium (broth). To accommodate the addition ofmedium, a fedbatch culture is started in a volume much lower that thefull capacity of the bioreactor (e.g., approximately 40% to 50% of themaximum volume).

Some methods of the present disclosure are directed to large-scaleproduction of RNA (e.g., ssRNA or dsRNA). For large-scale productionmethods, engineered cells may be grown in liquid culture medium in avolume of 5 liters (L) to 250,000 L, or more. In some embodiments,engineered cells may be grown in liquid culture medium in a volume ofgreater than (or equal to) 10 L, 100 L, 1000 L, 10000 L, or 100000 L. Insome embodiments, engineered cells are grown in liquid culture medium ina volume of 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L,100 L, 500 L, 1000 L, 5000 L, 10000 L, 100000 L, 150000 L, 200000 L,250000 L, or more. In some embodiments, engineered cells may be grown inliquid culture medium in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 Lto 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L. In someembodiments, engineered cells may be grown in liquid culture medium in avolume of 100 L to 300000 L, 100 L to 200000 L, or 100 L to 100000 L.

Typically, culturing of engineered cells is followed by lysing thecells. “Lysing” refers to the process by which cells are broken down,for example, by viral, enzymatic, mechanical, or osmotic mechanisms. A“cell lysate” refers to a fluid containing the contents of lysed cells(e.g., lysed engineered cells), including, for example, organelles,membrane lipids, proteins, nucleic acids and inverted membrane vesicles.Cell lysates of the present disclosure may be produced by lysing anypopulation of engineered cells, as provided herein.

Methods of cell lysis, referred to as “lysing,” are known in the art,any of which may be used in accordance with the present disclosure. Suchcell lysis methods include, without limitation, physical lysis such ashomogenization.

Cell lysis can disturb carefully controlled cellular environments,resulting in protein degradation and modification by unregulatedendogenous proteases and phosphatases. Thus, in some embodiments,protease inhibitors and/or phosphatase inhibitors may be added to thecell lysate or cells before lysis, or these activities may be removed byheat inactivation, gene inactivation, or protease targeting.

Cell lysates, in some embodiments, may be combined with at least onenutrient. For example, cell lysates may be combined with Na₂HPO₄,KH₂PO₄, NH₄Cl, NaCl, MgSO₄, CaCl₂. Examples of other nutrients include,without limitation, magnesium sulfate, magnesium chloride, magnesiumorotate, magnesium citrate, potassium phosphate monobasic, potassiumphosphate dibasic, potassium phosphate tribasic, sodium phosphatemonobasic, sodium phosphate dibasic, sodium phosphate tribasic, ammoniumphosphate monobasic, ammonium phosphate dibasic, ammonium sulfate,ammonium chloride, and ammonium hydroxide.

Cell lysates, in some embodiments, may be combined with at least onecofactor. For example, cell lysates may be combined with adenosinediphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adeninedinucleotide (NAD+), or other non-protein chemical compounds requiredfor activity of an enzyme (e.g., inorganic ions and coenzymes).

In some embodiments, cell lysates are incubated under conditions thatresult in RNA depolymerization. In some embodiments, cell lysates areincubated under conditions that result in production of ssRNA or dsRNA.

The volume of cell lysate used for a single reaction may vary. In someembodiments, the volume of a cell lysate is 0.001 to 250 m³. Forexample, the volume of a cell lysate may be 0.001 m³, 0.01 m³, 0.1 m³, 1m³, 5 m³, 10 m³, 15 m³, 20 m³, 25 m³, 30 m³, 35 m³, 40 m³, 45 m³, 50 m³,55 m³, 60 m³, 65 m³, 70 m³, 75 m³, 80 m³, 85 m³, 90 m³, 95 m, 100 m³,105 m³, 110 m³, 115 m³, 120 m³, 125 m³, 130 m³, 135 m³, 140 m³, 145 m³,150 m, 155 m³, 160 m³, 165 m³, 170 m³, 175 m³, 180 m³, 185 m³, 190 m³,195 m³, 200 m³, 205 m, 210 m³, 215 m³, 220 m³, 225 m³, 230 m³, 235 m³,240 m³, 245 m³, or 250 m³. In some embodiments, the volume of a celllysate is 25 m³ to 250 m³, 50 m³ to 250 m³, or 100 m³ to 250 m³.

Downstream Processing

The methods and systems provided herein, in some embodiments, yield RNA(e.g., dsRNA, ssRNA) product at a concentration of 1-50 g/L (e.g., 30,35, 40, 45, or 50 g/L). Downstream processing increases purity to asmuch as 99% (e.g., 75, 80, 85, 90, 95, 96, 97, 98, or 99%) dsRNA byweight. An example of downstream processing is shown in FIG. 4, startingwith the addition of a protein precipitating agent (e.g., ammoniumacetate) followed by disc-stack centrifugation (DSC) to remove protein,lipids, and some DNA from the product stream. Ultrafiltration is thenimplemented to remove salts and volume. Addition of lithium chloride tothe product stream leads to precipitation of the RNA product, which issubsequently separated from the bulk liquid using disc stackcentrifugation, for example, yielding an ˜80% purity RNA product stream.Further chromatographic polishing yield a ˜99% pure product.

ADDITIONAL EMBODIMENTS

Additional embodiments of the present disclosure are encompassed by thefollowing numbered paragraphs 1-46:

1. A cell-free method of biosynthesizing ribonucleic acid (RNA), themethod comprising:

(a) lysing cultured engineered cells that comprise RNA, an enzyme thatdepolymerizes RNA, thermostable kinases, a thermostable RNA polymerase,thereby producing a cell lysate;

(b) incubating the cell lysate produced in step (a) under conditionsthat result in depolymerization of RNA, thereby producing a cell lysatethat comprises nucleoside monophosphates;

(c) heating the cell lysate produced in step (b) to a temperature thatinactivates endogenous nucleases and phosphatases without inactivatingthe thermostable kinases and thermostable RNA polymerase, therebyproducing a cell lysate that comprises heat-inactivated nucleases andphosphatases; and

(d) incubating the cell lysate produced in (c) in the presence of anenergy source and an engineered DNA template containing a promoteroperably linked to a nucleotide sequence encoding a RNA of interest,under conditions that result in production of nucleoside triphosphatesand polymerization of the nucleoside triphosphates, thereby producing acell lysate that comprises the RNA of interest.

2. The method of paragraph 1, wherein the energy source ispolyphosphate, polyphosphate kinase, or both polyphosphate andpolyphosphate kinase.3. The method of paragraph 1 or 2, wherein the cultured engineered cellscomprise the engineered DNA template.4. The method of paragraph 1 or 2, wherein the engineered DNA templateis added to the cell lysate of step (d).5. The method of any one of paragraphs 1-4, wherein an ATP regenerationsystem is added to the cell lysate of step (d).6. The method of paragraph 1, wherein the cultured engineered cellsfurther comprise a thermostable polyphosphate kinase.7. The method of any one of paragraphs 1-6, wherein the RNA of theengineered cells of step (a) is endogenous RNA.8. The method of any one of paragraphs 1-7, wherein the RNA comprisesribosomal RNA, messenger RNA, transfer RNA, or a combination thereof.9. The method of any one of paragraphs 1-8, wherein the culturedengineered cells comprise at least two enzymes that depolymerize RNA.10. The method of any one of paragraphs 1-9, wherein the enzyme thatdepolymerizes RNA is selected from the group consisting of: S1 nuclease,Nuclease P1, RNase II, RNase III, RNase R, RNase JI, NucA, PNPase, RNaseT, RNase E, RNaseG and combinations thereof.11. The method of paragraph 10, wherein the enzyme that depolymerizesRNA is Nuclease P1.12. The method of any one of paragraphs 1-11, wherein the cell lysate ofstep (b) comprises a Mg²⁺⁻ chelating agent.13. The method of paragraph 12, wherein the Mg²⁺⁻ chelating agent isethylenediaminetetraacetic acid (EDTA).14. The method of paragraph 13, wherein the concentration of the EDTA is0.1 mM to 25 mM.15. The method of paragraph 14, wherein the concentration of the EDTA is8 mM.16. The method of any one of paragraphs 1-15, wherein the thermostablekinases comprise thermostable nucleoside monophosphate kinases.17. The method of paragraph 16, wherein the thermostable nucleosidemonophosphate kinases are selected from the group consisting ofthermostable uridylate kinases, thermostable cytidylate kinases,thermostable guanylate kinases, and thermostable adenylate kinases.18. The method of paragraph 17, wherein the stable nucleosidemonophosphate kinases a selected from the group consisting of athermostable Pyrococcus furiosus uridylate kinase encoded by a pyrH gene(PfPyrH), a thermostable Thermus thermophilus adenylate kinase encodedby a adk gene (TthAdk), a thermostable Thermus thermophilus cytidylatekinase encoded by a cmk gene (TthCmk), and a thermostable Pyrococcusfuriosus guanylate kinase encoded by a gmk gene (PfGmk).19. The method of any one of paragraphs 1-18, wherein the thermostablekinases comprise thermostable nucleoside diphosphate kinases.20. The method of paragraph 19, wherein the thermostable nucleosidediphosphate kinases are selected from the group consisting ofthermostable nucleoside phosphate kinases, thermostable pyruvatekinases, and thermostable polyphosphate kinases.21. The method of paragraph 20, wherein at least one of the thermostablenucleoside diphosphate kinases is a thermostable Aquifex aeolicus enzymeencoded by a ndk gene.22. The method of any one of paragraphs 1-21, wherein the cells comprisea thermostable nucleoside monophosphate kinase and a thermostablenucleoside diphosphate kinase.23. The method of any one of paragraphs 1-22, wherein the culturedengineered cells comprise thermostable uridylate kinase, thermostablecytidylate kinase, thermostable guanylate kinase, thermostable adenylatekinase, and thermostable polyphosphate kinase.24. The method of any one of paragraphs 1-23, wherein the thermostableRNA polymerase is a thermostable DNA-dependent RNA polymerase.25. The method of paragraph 24, wherein the DNA-dependent RNA polymeraseis selected from the group consisting of thermostable T7 RNApolymerases, thermostable SP6 RNA polymerases, and thermostable T3 RNApolymerases.26. The method of paragraph 25, wherein the DNA-dependent RNA polymeraseis a thermostable T7 RNA polymerase.27. The method of any one of paragraphs 1-26, wherein the temperature instep (c) is at least 50° C.28. The method of paragraph 27, wherein the temperature in step (c) isat 50° C.-80° C.29. The method of any one of paragraphs 1-28, wherein step (c) comprisesheating the cell lysate for at least 15 minutes.30. The method of any one of paragraphs 1-29, wherein step (c) comprisesheating the cell lysate to a temperature of at least 65° C. for 15minutes.31. The method of any one of paragraphs 1-30, wherein the nucleosidetriphosphates in step (d) are produced at a rate of 15-30 mM/hour.32. The method of any one of paragraphs 1-31, wherein the RNA ofinterest produced in step (d) is double-stranded RNA.33. The method of any one of paragraphs 1-32, wherein the RNA ofinterest is produced in step (d) a RNA interference molecule.34. The method of any one of paragraphs 1-33, wherein the RNA ofinterest produced in step (d) is an mRNA containing complementarydomains linked by a hinged domain.35. The method of any one of paragraphs 1-34, wherein the RNA ofinterest produced in step (d) is produced at a concentration of at least4 g/L, at least 6 g/L, at least 6 g/L, or at least 10 g/L.36. The method of paragraph 35 further comprising purifying thedouble-stranded RNA.37. The method of paragraph 36, wherein the purifying step comprisescombining the cell lysate of step (d) with a protein precipitating agentand removing precipitated protein, lipids, and DNA.38. A cell lysate produced by the method of any one of paragraphs 1-37.39. An engineered cell comprising RNA, an enzyme that depolymerizes RNA,a thermostable kinase and a thermostable RNA polymerase.40. The engineered cell of paragraph 39 further comprising an engineeredDNA template containing a promoter operably linked to a nucleotidesequence encoding a RNA of interest.41. A population of engineered cells of paragraph 39 or 40.42. A method, comprising:

maintaining in cell culture media engineered cells of paragraph 39.

43. The method of paragraph 42 further comprising lysing the culturedengineered cells to produce a cell lysate.44. The method of paragraph 43 further comprising incubating the celllysate under conditions that result in depolymerization of RNA toproduce a cell lysate that comprises nucleoside monophosphates.45. The method of paragraph 44 further comprising heating the celllysate to a temperature that inactivates endogenous nucleases andphosphatases without inactivating the thermostable kinases andthermostable RNA polymerase to produce a cell lysate that comprisesheat-inactivated nucleases and phosphatases.46. The method of paragraph 45 further comprising incubating the celllysate that comprises heat-inactivated nucleases and phosphatases in thepresence of an energy source and an engineered DNA template containing apromoter operably linked to a nucleotide sequence encoding a RNA ofinterest, under conditions that result in production of nucleosidetriphosphates and polymerization of the nucleoside triphosphates, toproduce a cell lysate that comprises the RNA of interest.

EXAMPLES Example 1 Nuclease Downselection

To identify the optimal nuclease(s) for digesting lysate RNA, a seriesof screening experiments were performed using commercially-availableenzymes chosen based on their ability to generate 5′-NMPs oroligonucleotides. The activity of these enzymes was first determinedusing purified E. coli RNA and reaction conditions recommended by themanufacturer, where RNA depolymerization was monitored by the release ofacid-soluble nucleotides. Under these conditions, four nucleasesdemonstrated depolymerization activity over background. Theendonucleases Benzonase and RNase A, which served as positive controls,yielded immediate conversion of RNA to acid-soluble nucleotides (FIG.5A). Treatment of RNA with the exonucleases P1 and RNase R yielded atime-dependent conversion of RNA to acid-soluble nucleotides, with RNaseR reaching nearly 100% depolymerization in 2 hours. The remainingnucleases (terminator exonuclease, RNase III and RNase T) did notproduce detectable depolymerization in this assay. Subsequent analysesby LC-MS revealed NMP liberation in samples treated with RNase R andNuclease P1, but not Benzonase or RNase A (FIG. 5B). These resultssuggest that RNase R and Nuclease P1 may be suitable for depolymerizinglysate RNA into 5′-NMPs. RNase R was chosen for further study forseveral reasons, including its lack of DNAse activity, its ability todegrade dsRNA and structured RNA, and its processive 3′→5′ exonucleaseactivity.

RNA Depolymerization in Lysates

RNase R was then tested for its ability to depolymerize endogenous RNAin bacterial lysates. In these experiments, purified RNase R (0.5 mg/mLfinal concentration) was added to lysates (50% final concentration), andfree nucleotides were quantified by UPLC. A representative experiment isshown in FIG. 6. Adding purified RNase R to lysates resulted in therapid release of 5′-NMPs from lysate RNA, with maximal NMP liberationafter 5-10 minutes. After this initial period of rapid depolymerization,NMP concentrations stabilized, then began to slowly decline. EndogenousRNase activity also resulted in 5′-NMP liberation, albeit at much lowerrates. Importantly, RNase R addition did not increase the rate of 2′ or3′ NMP liberation from RNA, consistent with its known mechanism ofaction. Across multiple independent experiments, addition of RNase R tolysates resulted in the conversion of 68% of lysate RNA to 5′-NMPs in5-10 minutes at rates in excess of 200 mM/hr.

To assess the toxicity of RNase R expression, two bacterial strains wereconstructed. One strain included the base strain (GL16-170) transformedwith an empty protein expression vector, and the other included GL16-170transformed with the same protein expression vector encoding RNase R.Both strains were grown under batch conditions in 1 L bioreactors,induced at OD600=20, and harvested before glucose exhaustion. Inductionyielded strong expression of RNase R (FIG. 7A) with no detectable changein growth rate (FIG. 7B). Upon lysis and 50% dilution, the strainexpressing RNase R exhibited rapid depolymerization of RNA intoacid-soluble nucleotides (FIG. 7C), indicating that overexpressed RNaseR was functional. Notably, additional RNase R activity, which wassupplied by adding purified enzyme to the reaction, did not increaserates of depolymerization or yields of acid-soluble nucleotides,suggesting that overexpressed RNase R is fully active upon lysis anddilution.

Next, the activity of overexpressed RNase R was assessed in high-densitylysates. Mg²⁺, which is known to stabilize ribosome structure andprotect rRNA from nucleases, is also required (in low amounts) for RNaseR activity. Therefore, depolymerization rates were measured in thepresence of varying concentrations of EDTA (FIG. 8). Lysates from theempty vector strain exhibited relatively slow depolymerization ratesthat were insensitive to EDTA, while lysates with overexpressed RNase Rexhibited higher rates of depolymerization with increasing EDTAconcentration, with maximal rates at 8 mM EDTA. Above 8 mM,depolymerization rates decreased, likely due to inactivation ofMg²⁺-dependent RNase R. Taken together, these results suggest thatoverexpressed RNase R is non-toxic and can be activated upon lysis.

NMP Stability in Lysates

After RNA depolymerization, the resulting NMP pool is progressivelyphosphorylated to NTPs before polymerization into dsRNA. Deleteriousenzymatic activities, such as NMP degradation into nucleosides andsubsequent hydrolysis into sugars and bases, negatively impact dsRNAyields. Therefore, the stability of individual NMPs was assessed inlysates. Stability assessments were performed by addingisotopically-labeled “heavy” NMPs (hAMP, hCMP, hUMP, and hGMP) tolysates, and quantifying abundance over time using LC-MS (Figured 9A-9D,solid lines). In contrast to hAMP, which is relatively stable, hCMP,hUMP, and hGMP are actively degraded by the lysate, with approximatehalf-lives (t_(1/2)) of 1 hour, 30 minutes, and 20 minutes,respectively.

One pathway for metabolism of NMPs is dephosphorylation intonucleosides. To assess whether dephosphorylation was contributing to NMPdegradation, stability assessments were repeated with the addition ofinexpensive phosphatase inhibitors. Increased concentrations ofphosphate (PO₄, 150 mM), as well as the structural mimic orthovandate(VO₄, 10 mM) were pre-incubated with lysates before hNMP addition.Increasing phosphate concentration stabilized hUMP (t_(1/2)≈60 minutes),while minimally affecting hCMP and hGMP (FIGS. 9A-9D, dashed lines). Incontrast, orthovanadate stabilized hCMP (t_(1/2)>>60 minutes), hUMP(t_(1/2)≈60 minutes), and hGMP (t_(1/2)≈45 minutes) (FIGS. 9A-9D, dottedlines). Taken together, these stability assessments define an overallNMP degradation rate of 13 mM/hr in lysates, with 70% ofexogenously-added NMPs present after 15 minutes. Even in the absence ofphosphatase inhibitors, the relatively low rate of hNMP consumption (13mM/hr) compared to the rate of RNase R-dependent depolymerization (>200mM/hr), suggested that NMPs released from RNA should be available forpolymerization into dsRNA.

Development of Heat Inactivation

To stabilize NMPs, as well as NDPs, NTPs, and dsRNA, a heat inactivationprotocol was developed. The objective was to identify the lowesttemperature and shortest incubation time that would eliminate nucleotideand RNA degradation activities in lysates. To assess the efficacy ofheat inactivation, NTP consumption rates (at 37° C.) were comparedacross heat-inactivated lysates by LC-MS, where the time and temperatureof heat inactivation was varied. Before heat inactivation, lysatesconsumed NTPs at approximately 120 mM/hr (FIG. 10). Temperatures below70° C. did not affect NTPase activity, while incubation at 70° C.produced a time-dependent decrease in NTPase activity. Completeinhibition of NTPase activity occurred after a 15 minute incubation at70° C. (FIG. 10).

Next, these conditions were evaluated for their ability to stabilizeNMPs and dsRNA in lysates. To evaluate the effects of heat inactivationon NMP degradation, lysates were treated with exogenous RNase R torelease RNA, then subjected to heat inactivation at 70° C.Post-inactivation, the temperature was lowered to 37° C., and thereaction incubated for an additional 60 minutes. As shown in FIG. 11A,treatment with RNase R for 5 minutes rapidly depolymerized RNA. Afterheat inactivation and incubation at 37° C., NMP concentrations wereunchanged, suggesting that the chosen heat-inactivation conditionsrendered NMPs stable.

Finally, these conditions were evaluated for their ability to stabilizethe reactants and products of an in vitro transcription reaction(including NTPs and dsRNA) in lysates. First, lysates were pre-incubatedat elevated temperature for 15 minutes. Then, the temperature waslowered to 37° C. and transcription reactants (including exogenous NTPs,DNA template, and purified T7 RNA polymerase) were added. As shown inFIG. 11B, transcription reactions in lysates that were heat inactivatedat >70° C. yielded RNA products qualitatively similar to the positivecontrol reaction (performed in buffer). No RNA product was apparent inthe reaction performed at 60° C.

Taken together, these results suggest that a 70° C. incubation for 15minutes is sufficient to stabilize NMPs, NTPs, and dsRNA in cell-freereactions.

Selection and Evaluation of Thermostable Kinases

After heat-inactivation, a series of kinase activities are required tosequentially phosphorylate 5′-NMPs liberated from RNA to NTPs that canbe polymerized to form dsRNA. These kinases, which use high-energyphosphate groups from ATP to phosphorylate NMPs and NDPs, must besufficiently thermostable to remain active following high-temperatureincubation, as well as sufficiently active to produce NTPs at high rates(21 mM/hr NTPs for 1 g dsRNA/L/hr). Enzymes from thermophilic organismswere chosen for evaluation (Table 9) based on literature reports ofsuccessful expression in E. coli and biochemical characterization of therecombinant enzymes.

TABLE 9 Origins of thermostable kinases tested for each class ofactivity. Enzyme class Organism Prefix NMP kinase Escherichia coli EcThermus thermophilus Tth Pyrococcus furiosus Pf Thermosynechococcus Teelongatus Thermotoga maritima Tm NDP kinase Escherichia coli Ec Thermusthermophilus Tth Aquifex aeolicus Aa Polyphosphate kinase Escherichiacoli Ec Thermus thermophilus Tth Thermosynechococcus Te elongatus

To evaluate the suitability of these enzymes for cell-free production ofdsRNA, enzymes were cloned into an E. coli protein expression vectorwith an N-terminal hexahistidine tag, overexpressed, and purified usingimmobilized metal affinity chromatography (IMAC). Activities of thepurified enzymes were first quantified using luciferase-coupled assays,where the consumption of ATP served as a proxy for NMP and NDPphosphorylation. Assays were performed at a range of incubationtemperatures to determine the optimal reaction temperature for eachenzyme.

Expression of UMP kinases (encoded by the pyrH gene) from T.thermophilus and E. coli yielded insoluble protein under the testedinduction and purification conditions. Purified PyrH from P. furiosusexhibited a specific activity of approximately 2 μmol/min/mg proteinthat was largely temperature-independent (FIG. 12). Based on thisspecific activity, a high-density cell lysate (90 g dcw/L) expressingPfPyrH at 1% total protein would, in the presence of excess ATP,phosphorylate UMP at rates in excess of 50 mM/hr (Table 10). As dsRNAproduction at 1 g/L/hr requires approximately 5.25 mM/hr of UMP kinaseactivity, PfPyrH was chosen for further evaluation. Table 10 showspredicted PyrH rates in high-density (90 g dcw/L) lysates, assumingPfPyrH constitutes 1% of total lysate protein.

TABLE 10 Predicted PyrH reaction rates Temperature (° C.) Predicted Rate(mM/hr) 50 51.0 60 52.4 70 51.8 80 59.7

Expression of AMP kinases (encoded by the adk gene) from E. coli and T.thermophilus yielded soluble recombinant protein, while theThermosynechococcus enzyme was insoluble under the tested expression andpurification conditions. The purified E. coli enzyme was active at 37°C. and 50° C., but exhibited no detectable activity at highertemperatures (FIG. 13). The T. thermophilus enzyme exhibited higherspecific activity than the E. coli enzyme at all tested temperatures,with an optimal activity of nearly 1 mM/min per mg enzyme at 70° C. Thisactivity, when the enzyme is expressed at 0.01% of total protein in ahigh-density lysate, translates to an expected rate in excess of 250mM/hr of AMP phosphorylation in lysates (Table 11). As approximately5.25 mM/hr of AMP kinase activity is required to synthesize 1 g/L/hrdsRNA, TthAdk was chosen for further study. Table 11 shows predicted Adkreaction rates in high-density (90 g dcw/L) lysates, assuming TthAdkconstitutes 0.01% of total lysate protein.

TABLE 11 Predicted Adk reaction rates Temperature (° C.) Predicted Rate(mM/hr) 37 n.d. 50 86.4 60 199.5 70 253.8 80 106.1

Expression of CMP kinases (encoded by the cmk gene) from E. coli and P.furiosus yielded insoluble protein, while expression of the T.thermophilus enzyme yielded soluble protein under the tested conditions.T. thermophilus CMP kinase exhibited activity largely independent oftemperature, although enzyme activity decreased slightly at temperaturesabove 60° C. (FIG. 14). Based on these results, expression of TthCmk inhigh-density lysates at 0.02% of total protein would yield CMP kinaseactivities of 30-45 mM/hr (depending on temperature, Table 12), well inexcess of the 5.25 mM/hr target. Therefore, TthCmk was chosen forfurther evaluation. Table 12 shows predicted Cmk reaction rates inhigh-density (90 g dcw/L) lysates, assuming TthCmk constitutes 0.02% oftotal lysate protein.

TABLE 12 Predicted Cmk reaction rates Temperature (° C.) Predicted Rate(mM/hr) 37 44.8 50 44.0 60 40.8 70 30.2 80 32.2

In contrast to the tested CMP kinases, expression of GMP kinases from E.coli, T. thermophilus, and T. maritima yielded soluble recombinantprotein. The E. coli enzyme had the highest tested specific activity at37° C., but was less active at higher temperatures (FIG. 14). While theT. thermophilus and T. maritima enzymes both exhibited optimal activityat higher temperatures, the T. maritima enzyme was more active at alltested temperatures. Based on the measured specific activities of TmGmk,expression in a high-density cell lysate at 0.1% of total protein wouldyield an expected rate in excess of 60 mM/hr at 70° C. in the presenceof excess ATP, compared to a target rate of 5.25 mM/hr (Table 13).Therefore, TmGmk was chosen for further evaluation. Table 13 showspredicted Gmk reaction rates in high-density (90 g dcw/L) lysates,assuming TmGmk constitutes 0.1% of total lysate protein.

TABLE 13 Predicted Gmk reaction rates Temperature (° C.) Predicted Rate(mM/hr) 37 41.8 50 42.8 60 55.2 70 62.0 80 52.4

Unlike the NMP kinases, which are largely specific for a singlesubstrate, NDP kinase phosphorylates ADP, CDP, UDP, and GDP. To compareNDP kinases, enzymes from the thermophiles T. thermophilus and A.aeolicus were cloned and expressed in E. coli. While the T. thermophilusenzyme was insoluble under the tested conditions, expression of the A.aeolicus enzyme yielded soluble protein. Activity measurements in aluciferase assay using ATP and GDP as substrates revealed that AaNdk ishighly active across a broad range of temperatures, with a temperatureoptimum of 50° C. (FIG. 16). Comparison across substrates revealedspecific activities in excess of 100 μmol/min/mg at 50° C. for eachnucleotide, confirming that AaNdk could phosphorylate multiple NDPsubstrates (Table 14). Based on these measurements, expression of AaNdkin high density lysates at 0.01% total protein translates to UDP kinaserates well in excess of 60 mM/hr. Given that 21 mM/hr of NDP kinaseactivity is required to support 1 g/L/hr dsRNA synthesis, AaNdk waschosen for further evaluation. Table 14 shows that at 50° C., AaNdk ishighly active with CDP, UDP, and GDP substrates.

TABLE 14 AaNdk specific activity by substrate. Substrate SpecificActivity (μmol/min/mg) CDP 259.6 UDP 110.4 GDP 217

Polyphosphate kinase (Ppk) reversibly transfers high-energy phosphategroups between polymeric phosphate chains and adenosine nucleotides.Multiple Ppk enzymes, belonging to the Type I family of polyphosphatekinases, were evaluated for activity, including enzymes from E. coli aswell as the thermophilic organisms T. thermophilus andThermosynechococcus elongatus. These enzymes were selected for testingas they belonged to the well-characterized Type I family or hadpreviously been shown to be active. Expression and purification of Ppkenzymes yielded soluble protein, which was then tested for activity in aluciferase assay system using ADP and sodium hexametaphosphate assubstrates. As shown in FIG. 17, the specific activity of all tested Ppkenzymes was low relative to other kinase activities. The E. coli Ppk hadthe highest specific activity at lower temperatures (up to 60° C.),while the Thermosynechococcus enzyme had the highest activity at 70° C.Additionally, incubation of the E. coli enzyme under heat inactivationconditions (70° C. for 15 minutes) led to irreversible inactivation (notshown). Based on the specific activity of the Thermosynechococcusenzyme, expression in a high density lysate at 2% of total protein wouldlead to an expected rate of 42 mM/hr at 70° C. (Table 15), matching therequired ATP production rate to support 1 g/L/hr dsRNA synthesis.However, cell-free dsRNA synthesis at lower temperatures (e.g., 50° C.)would require higher expression of TePpk (in excess of 4% totalprotein). Table 15 shows predicted Ppk reaction rates in high-density(90 g dcw/L) lysates, assuming TePpk constitutes 2% of total lysateprotein.

TABLE 15 Predicted PPK1 reaction rates Temperature Predicted Rate (° C.)(mM/hr) 37 6.5 50 18.9 60 38.9 70 42.1 80 0

After evaluating each enzymatic activity in a purified system, enzymeswere tested for activity in heat-inactivated lysates. Lysates expressingindividual kinases were pre-incubated at 70° C. for 15 minutes beforesubstrates were added. As in the purified reactions, ATP consumption(for NMP and NDP kinases) or ATP production (for polyphosphate kinase)were quantified using a luciferase assay kit. As shown in Table 16below, rates of NMP and NDP phosphorylation were well in excess oftargets.

TABLE 16 Kinase activities in heat-inactivated lysates Rate TargetEnzyme Reaction (mM/hr) (mM/hr) PfPyrH UMP + ATP ⇄ UDP + 151 5.25 ADP +P_(i) TthAdk AMP +ATP ⇄ 2 ADP + 199 5.25 2 P_(i) TthCmk CMP +ATP ⇄ CDP +183 5.25 ADP + P_(i) PfGmk GMP +ATP ⇄ GDP + 168 5.25 ADP + P_(i) AaNdkNDP +ATP ⇄ NTP + 163 21 ADP + P_(i) TePpk ADP +Poly-P_(n) ⇄ ATP + 2.7 42Poly-P_(n−1)

After confirming that individual kinases were sufficiently active inlysates (with the exception of Ppk), kinases were evaluated in amulti-enzyme system for their ability to convert NMPs to NTPs. In thesestudies, equal volumes of lysates expressing individual kinases (5) werecombined, heat-inactivated, then assayed for ATP-dependent production ofNTPs from an equimolar mix of NMPs by LC-MS. As shown in Table 17,overall NTP production rates exceeded 24 mM/hr for UTP, CTP, and GTP,suggesting that a simple mixture of lysates, without any optimization ofreaction conditions, could provide NTPs at sufficient rates to supportsynthesis of 1 g/L/hr dsRNA, in the presence of adequate ATP.

TABLE 17 Production of NTPs in a heat-inactivated 1:1 mixture of lysatesexpressing individual kinase activities, using an equimolar mixture ofNMPs as substrates and ATP as a high-energy phosphate donor Pathway Rate(mM/hr) UMP → UTP 3.24 CMP → CTP 9.24 GMP → GTP 11.8 AMP → ATP n.d NMPs→ NTPs >24.3

RNA Polymerase Downselection

After depolymerization of RNA into NMPs and phosphorylation of NMPs totheir corresponding NTPs, a RNA polymerase is required to convert NTPsinto the dsRNA product. RNA polymerase from the bacteriophage T7 is anattractive enzyme for use in a recombinant system for several reasons.T7 RNA polymerase includes a single subunit (unlike many RNA polymerasesfrom Bacteria and Eukarya) and has been extensively characterized bybiochemical and molecular biology studies. Additionally, multiple T7 RNApolymerase mutants have been described that confer improvedthermostability (see Table 18).

TABLE 18 List of T7 RNA polymerases evaluated for activity andthermostability Enzyme Name Source Rationale MegaScript T7 InvitrogenComponent of high-yield transcription kit T7 RNA Polymerase New EnglandBiolabs Control ThermoT7 Toyobo Life Sciences Claimed activity RNAPolymerase at 50° C. «TT7» JP4399684 (Toyobo) T7 RNA EP2377928 (Roche)Claimed activity Polymerase (LVI) at 50° C. T7 RNA EP1261696(bioMérieux) Claimed activity Polymerase (PPIY) at 50° C. T7 RNAPolymerase Combination of Mutations potentially (LPPVIIY) LVI & PPIYsynergistic

First, the activities of commercially-available T7 RNA polymerases wereevaluated using duplex DNA template (e.g. FIG. 3B) and a 37° C. reactiontemperature. The production of RNA was quantified over time using theQuant-iT RNA Kit (Broad Range) (Thermo Fisher Scientific). Underconditions recommended by each manufacturer, the ThermoT7 and MegaScriptenzymes were highly active, while the NEB enzyme displayed significantlylower activity (FIG. 18).

Next, the LVI mutant of T7 RNA polymerase was cloned with an N-terminalhexahistidine tag, expressed in E. coli, and purified using IMAC.

Next, activities of the MegaScript and ThermoT7 polymerases were testedalongside the LVI mutant polymerase in dilute heat-inactivated lysates(35% final lysate concentration) under standardized reaction conditions(FIG. 19). As in a purified system, ThermoT7 exhibits higher specificactivity at 37° C. (3.1 nmol RNA/min/mg protein) than the MegaScriptenzyme. The LVI mutant had the lowest specific activity (0.33nmol/min/mg) of the three enzymes tested. When tested under otherwiseidentical conditions at 50° C., neither the MegaScript enzyme nor theLVI mutant exhibited any detectable activity. In contrast, activity ofThermoT7 was higher than at 37° C. With approximately 4% of totalprotein in the assay as ThermoT7 polymerase, RNA synthesis ratesexceeded 11 g/L/hr at 50° C. with duplex DNA template and ThermoT7polymerase (FIG. 19). ThermoT7 was then selected for furthercharacterization.

After confirming the activity of ThermoT7 at 50° C. in diluteheat-inactivated lysate, the activity of ThermoT7 was investigated underconditions representative of cell-free dsRNA production at scale. Inaddition to increased concentrations of lysate, reactions at scale mayinclude precipitated lysate components (e.g., protein) that arise fromthe heat inactivation process. To assess the performance of ThermoT7under these conditions, RNA polymerization was quantified inheat-inactivated high-density lysates (68% final lysate concentration)with and without clarification to remove precipitated proteins afterheat inactivation (FIG. 20). RNA synthesis rates were significantlyhigher in matrix than in buffer, with the highest rates occurring inheat-inactivated matrix that had been clarified by centrifugation. Inunclarified reactions, overall RNA synthesis rates (over a 2-hourreaction) were in excess of 2 g/L/hr with 1.4% of total protein in theassay as ThermoT7 polymerase.

Finally, the thermostability of ThermoT7 was tested at highertemperatures to evaluate compatibility with the heat inactivationconditions established earlier in this program. In these studies,ThermoT7 enzyme was pre-incubated at elevated temperature (50-70° C.)for varying lengths of time (0-15 minutes), and the remaining activityquantified at 37° C. As shown in FIG. 21, incubation at 50° C. iswell-tolerated by the enzyme, but higher temperatures lead to rapidirreversible inactivation of polymerase activity. Therefore, in theseexperiments, this particular Thermo T7 was not compatible withheat-inactivation at 50-70° C. Thus, in some instances, purifiedThermoT7 enzyme may be added to cell-free reactions following a thermalinactivation, or an alternate T7 RNA polymerase mutant may be used,having sufficient half-life at 70° C., for example.

Materials and Methods Nucleotide Analysis

Analysis of nucleotide monophosphates (AMP, CMP, UMP, and GMP) wasperformed by liquid chromatography coupled with mass spectrometry(LC-MS). Samples were separated using an Agilent 1100 series HPLCequipped with a ZIC-cHILIC column (2.1×20 mm, 3 μm i.d.) (Merck) at roomtemperature with a flow rate of 0.5 mL/min and a 2 injection volume.Mobile phases consisted of 20 mM ammonium acetate (A) and 20 mM ammoniumacetate in 90% acetonitrile (B). The separation method consisted of agradient from 15-50% (B) for 3.5 minutes, followed by 50% (B) for 1.5minutes, then 15% B for 3 minutes. Quantification was performed on anABSciex API 3200 mass spectrometer using electrospray ionization(capillary voltage: −3000V, temperature: 600° C., desolvation gas: 20psi) in multiple reaction monitoring (MRM) mode. Analysis of nucleotidemonophosphate, diphosphate, and triphosphate species (NMPs, NDPs, andNTPs) used the method described above with the following separationgradient: 15% B to 50% B for 3.5 minutes, followed by 50% B for 2.5 min,then 15% B for 4 minutes. Peak areas were compared to standard curvesconsisting of purified compounds (Sigma-Aldrich). For analysis ofsamples in lysates, standard curves were prepared in lysate backgroundsthat had been acid-quenched, clarified, pH-neutralized, and filtered asin the sample preparation steps described below.

Analysis of 2′-, 3′-, and 5′-NMPs was performed by liquid chromatographyusing an ACQUITY H-Class UPLC (Waters) equipped with an ACQUITY CSHfluoro-phenyl column (2.1×150 mm, 1.7 μm i.d.) (Waters) at 40° C. with aflow rate of 0.5 mL/min and a 0.5 μL injection volume. Mobile phasesconsisted of 10 mM ammonium acetate in 0.2% formic acid (A) and 10 mMammonium acetate in 95% acetonitrile, 0.2% formic acid (B). Theseparation method consisted of 1% B for 2.8 minutes, followed by agradient from 1%-30% B for 2.2 minutes, followed by 100% B for 7minutes, then 1% B for 3 minutes. Quantification was performed using anACQUITY UPLC PDA (Waters) at 260, 254, and 210 nm. Peak areas werecompared to standard curves consisting of purified compounds (purchasedfrom Sigma-Aldrich except for 2′ and 3′ CMP, UMP, and GMP which werepurchased from Biolog Life Science Institute). For analysis of samplesin lysates, standard curves were prepared in lysate backgrounds that hadbeen acid-quenched, clarified, pH-neutralized, and filtered as in thesample preparation steps described below.

Extraction and Purification of E. coli RNA

RNA was extracted and purified from high-density E. coli lysates(protein concentration: 40-50 mg/mL) according to established protocols(Mohanty, B. K., Giladi, H., Maples, V. F., & Kushner, S. R. (2008).Analysis of RNA decay, processing, and polyadenylation in Escherichiacoli and other prokaryotes. Methods in enzymology, 447, 3-29). For every400 μL of frozen E. coli lysate, 67 μL of 20 mM acetic acid was added toreduce RNase activity. Samples were thawed in a bead bath at 37° C.Immediately upon thawing, 400 μL of a 10% (w/v) solution oftrimethyl(tetradecyl)ammonium bromide (Sigma-Aldrich) was added. Theresulting suspensions were then clarified by centrifugation at 10,000×gin a microcentrifuge at 4° C., and the supernatant removed. Pellets wereresuspended in 1 mL of a 2M solution of lithium chloride (Sigma-Aldrich)in 35% ethanol. The suspensions were incubated at room temperature for 5minutes, then clarified by centrifugation at 15,000×g for 6 minutes at4° C. and the supernatants removed. Pellets were then resuspended in 1mL of a 2M solution of lithium chloride in water, and incubated at roomtemperature for 2 minutes before clarification at 15,000×g for 6minutes. Supernatants were then removed and the remaining pellets washedby resuspending in 70% ethanol and centrifuging at maximum speed(21,000×g) for 5 minutes at 4° C. Supernatants were then removed and thepellets were air-dried for 15 minutes at room temperature. Pellets werethen resuspended in 200 μL nuclease-free water, and incubated overnightat 4° C. to solubilize RNA. RNA solutions were clarified bycentrifugation (maximum speed for 5 minutes at 4° C.) and supernatantscontaining soluble RNA were transferred to sterile RNase-free tubes andstored at −20° C.

Nuclease Downselection

Nucleases were obtained from commercial sources as follows: Benzonaseand Nuclease P1 were obtained from Sigma-Aldrich, RNase R, Terminatorexonuclease, and RNase III were obtained from Epicentre, RNase A wasobtained from Thermo Fisher, and Exonuclease T was obtained from NewEngland BioLabs. For screening assays, 1 μL of each enzyme solution wasadded to 100 μL of 2× assay buffer (100 mM potassium phosphate pH 7.4,10 mM magnesium chloride, 1 mM zinc chloride), then combined with anequal volume of 1 mM RNA solution (˜340 ng/μL) at time t=0 and mixedwell. Reactions were incubated at 37° C. and periodically sampled bytransferring 20 μL to acid quench solution (180 μL of 0.2M sulfuricacid) on ice. After completion of the time course, quenched samples wereclarified by centrifugation at 3,000×g for 5 minutes at 4° C. 170 μL ofsupernatant from each sample was then transferred to a UV-transparent96-well half area plate (Corning) and acid-soluble nucleotides werequantified by absorbance at 260 nm using a microplate reader and anextinction coefficient of 10665 M⁻¹ cm⁻¹, estimated by averagingindividual extinction coefficients for each mononucleotide. Forsubsequent analysis by LC-MS, 45 μL clarified supernatant waspH-neutralized with 5 μL of 2.5 M potassium hydroxide. The totalnucleotide pool (i.e. 100% depolymerization) was determined by alkalinehydrolysis of RNA: RNA was combined with an equal volume of 0.2Mpotassium hydroxide, then heated to 99° C. for 20 minutes.Alkaline-hydrolyzed samples were then quenched and analyzed as describedabove.

Protein Expression and Purification

Recombinant proteins were cloned from synthetic DNA encoding therelevant gene along with a hexahistidine tag into pETDuet-1 (Novagen).Plasmids were transformed into E. coli T7Express (New England Biolabs),then grown in 1 L cultures using ZYM-505 media (Studier, F. W. (2005).Protein production by auto-induction in high-density shaking cultures.Protein expression and purification, 41(1), 207-234) supplemented with50 μg/mL carbenicillin. Expression was induced at A₆₀₀=0.6. For RNase Rand kinases, expression was induced with 0.1 mM IPTG, the temperaturelowered to 16° C., and the culture grown for 24 hours at 16° C. For T7RNA polymerase, expression was induced with 0.8 mM IPTG and the culturegrown for 3 hours at 37° C. Biomass was harvested by centrifugation andthe supernatant decanted before storing the cell pellets at −80° C. Cellpellets were thawed and lysed by resuspension into 4 volumes B-PERComplete (Thermo Fisher Scientific) supplemented with Benzonase (0.04μL/mL) and incubation with gentle agitation for 15 minutes at roomtemperature. Lysates were then clarified by centrifugation at 16,000×gfor 1 hour at 4° C. Proteins were purified by immobilized metal affinitychromatography using His GraviTrap columns (GE Healthcare) or HisTrap HPcolumns connected to an AKTAPrime Plus FPLC system (GE Healthcare). Forboth purification methods, columns were equilibrated inEquilibration/Wash buffer (50 mM phosphate buffer pH 7.4, 500 mM NaCl,20 mM imidazole), loaded with lysate, and then washed with 30 columnvolumes Equilibration/Wash Buffer. Proteins were eluted with ElutionBuffer (50 mM phosphate buffer pH 7.4, 500 mM NaCl, 500 mM imidazole).For purification of kinases, Equilibration/Wash and Elution buffers used50 mM Tris-HCl pH 7.5 instead of phosphate buffer. Elution fractionswere analyzed by SDS-PAGE and protein content quantified by BCA (ThermoFisher Scientific). Fractions were then combined and buffer exchanged bydialysis into 1000 volumes 2× Storage Buffer. For RNase R, 2× StorageBuffer consisted of 2×PBS supplemented with an additional 500 mM NaCl.For T7 RNA polymerase, 2× Storage Buffer consisted of 2×PBS. Forkinases, 2× Storage Buffer consisted of 100 mM Tris-HCl pH 7.0 with 100mM NaCl. After dialysis, proteins were mixed with an equal volume of100% glycerol (50% final concentration) and stored at −20° C.

Cell Lysate Preparation

E. coli strains GL16-170(BL21(DE3).t526pgi.Δedd.ΔtktB.ΔtolC_wt-7-E1.ΔmgsA*-F3.ΔappA*.Δamn*-F1.nagD(keio)::zeoR-1.ΔphoA*.t352BAA1644.ΔushA*-C4.rna::tolC-B04)and GL14-322(BL21(DE3).t526pgi.Δedd.ΔtktB.ΔtolC_wt-7-E1.ΔmgsA*-F3.ΔappA*.Δamn*-F1.nagD(keio)::zeoR-1.ΔphoA*.t352BAA1644.ΔushA::tolC-A01)were grown in Korz media in 10 L bioreactors until the end of batchphase, then harvested by centrifugation and frozen at −80° C. Pelletswere resuspended to 10% dry cell weight in 58.8 mM potassium phosphatedibasic and lysed using 2 passes through a PandaPLUS homogenizer (GEANiro Soavi) cooled to 4° C. at 15,000 psi. Lysates were clarified bycentrifugation at 16,000×g for 1 hour at 4° C. and protein content wasanalyzed by BCA assay (Thermo Fisher) before storage at −80° C.

Depolymerization of Lysate RNA with Exogenous RNase R

GL16-170 lysate (protein content 34.5 mg/mL) and RNase R solution (1mg/mL in 300 mM potassium phosphate buffer pH 7.4, 200 mM KCl, 2 mMMgCl₂) were pre-equilibrated at 2° C. before initiating the reaction. Attime t=0, 50 μL of E. coli lysate and 50 μL RNase R solution were mixedand the reaction initiated by transferring to a preheated 37° C. block.Reactions including deoxycholate were assembled as described above,except that lysates were premixed with 0.2 volumes of 5× sodiumdeoxycholate solutions in water and incubated at 2° C. for 15 minutesbefore initiation. After initiation, reactions were incubated at 37° C.and periodically sampled by transferring 10 μL to acid quench solution(90 of 0.2M sulfuric acid) on ice. After completion of the time course,quenched samples were clarified by centrifugation at 3,200×g for 10minutes at 2° C. Depolymerization was first quantified by absorbance ofacid-soluble nucleotides: 10 μL of quenched and clarified reactions wasadded to 160 μL of 0.2M sulfuric acid in a UV-transparent 96-well halfarea plate (Corning). Acid-soluble nucleotides were quantified byabsorbance at 260 nm using a microplate reader (see above).Depolymerization was also quantified by UPLC analysis of 5′, 2′, and 3′NMPs: 30 μL of each acid-quenched sample was pH-neutralized by adding 10of 1M KOH, then passed through a 0.2 μm filter before UPLC analysis. Thetotal nucleotide pool (i.e. 100% depolymerization) was determined byalkaline hydrolysis of lysate RNA: 50 lysate was combined with 150 μL of0.2M potassium hydroxide, then heated to 99° C. for 20 minutes.Alkaline-hydrolyzed samples were then quenched and analyzed as describedabove.

Depolymerization of RNA in Lysates with Overexpressed RNase R

E. coli strain GL16-170 was transformed with pETDuet-1 encoding the E.coli rnr gene with a C-terminal hexahistidine tag. This strain,alongside GL16-170 transformed with empty pETDuet-1, was grown in batchphase in Korz medium supplemented with 50 mg/L carbenicillin. Cultureswere induced with 0.8 mM IPTG at A₆₀₀=20 and supplemented with anadditional 10 g/L glucose at induction. One hour following induction,biomass was harvested by centrifugation and frozen. Lysates wereprepared from frozen biomass as described above (Protein concentrations:36.6 mg/mL for GL16-170 biomass with empty pETDuet-1; 53.2 mg/mL forGL16-170 with pETDuet-1 carrying cloned RNase R). Depolymerization indilute lysates was assessed as described above with 50% final lysateconcentration in the reaction. Depolymerization in concentrated lysateswas assessed by pre-incubating 9 volumes lysate with 1 volume 10×EDTAsolution for 5 minutes at 2° C. Reactions were then initiated bytransferring to a preheated 37° C. block and sampling as describedabove.

NMP Stability Assessment

Four volumes of GL14-322 lysate (protein concentration: 50.5 mg/mL) werecombined with one volume of phosphatase inhibitor solution (finalconcentrations of 50 mM potassium phosphate pH 7.4, 150 mM potassiumphosphate pH 7.4, or 10 mM sodium orthovanadate) on ice. An equimolarsolution of isotopically labeled NMPs (Adenosine-¹³C₁₀, ¹⁵N₅5′-monophosphate, Cytidine-¹⁵N₃-5′-monophosphate, monophosphate, andGuanosine-¹⁵N₅-5′-monophosphate [Sigma-Aldrich], 25 mM each) wasprepared in water. Lysates and NMPs were equilibrated to 37° C. for 10minutes before reactions were initiated. To initiate reactions, 90 μLlysate solution was added to 10 μL NMP solution, and the reactions mixedwell. Reactions were monitored by sampling at the indicated time points.During sampling, 12 μL of reaction mixture were transferred to 108 of0.2M sulfuric acid on ice. Quenched reactions were then clarified bycentrifugation, pH-neutralized, and filtered for LC-MS analysis asdescribed above.

Development of Heat Inactivation

GL14-322 lysate was aliquoted into 5 microcentrifuge tubes on ice, thentransferred to a heat block equilibrated at the desired heatinactivation temperature. At the indicated times, tubes were cooled onice, then clarified by centrifugation (21,000×g for 5 minutes) and thesupernatants harvested. Supernatants from heat-inactivated lysates,along with an equimolar mixture of NTPs (Sigma-Aldrich, 25 mM each) wereequilibrated at 37° C. for 10 minutes. At time t=0, 9 volumes ofheat-inactivated lysate supernatant were combined with 1 volume of NTPsolution, and the reaction monitored by sampling into acid quenchsolution, pH-neutralized, filtered, and analyzed by LC-MS as describedabove. For transcription reactions in lysates, 10 μL reactions wereperformed using the MegaScript T7 Transcription Kit (Thermo Fisher)following kit instructions, except for a reduced amount of enzyme mix(5% of final reaction volume), and including heat-inactivated lysatesupernatant (40% of final reaction volume). Positive control reactionswere performed in MegaScript reaction buffer alone, while negativecontrol reactions included lysate but omitted enzyme mix. Reactions wereanalyzed by agarose gel electrophoresis stained with SYBR Safe(Invitrogen) alongside the 1 kb DNA ladder (New England Biolabs).

Nucleotide Kinase Activity Assays

Nucleotide kinases were assayed at varying temperatures (37° C., 50° C.,60° C., 70° C., and 80° C.) in a buffer consisting of 50 mM Tris-HCl pH7.0, 4 mM MgSO₄, 4 mM ATP, and 4 mM of the corresponding NMP or NDP.Reaction buffer (1.2× concentrate) and enzyme solution (0.5 mg/mL) werepre-equilibrated at reaction temperature for 1 minute before reactionswere initiated. At time t=0, reactions were initiated by mixing 80reaction buffer with 20 μL enzyme. Reactions were monitored by samplingat the indicated time points. During sampling, 15 μL of reaction mixturewere transferred to 135 μL of 0.2M sulfuric acid on ice. Aftercompletion of the reaction, samples were pH-neutralized with 1M KOH asdescribed above, then diluted 1:10 in ice-cold water. ATP was quantifiedin each sample using the ATP Bioluminescent Assay Kit (Sigma-Aldrich),following kit instructions. For reactions in lysates, the above protocolwas modified as follows: lysates were aliquoted into individual reactiontubes, then heat-inactivated by incubating at 70° C. for 15 minutes.Reaction buffer (2× concentrate) and heat-inactivated lysates werepre-equilibrated at reaction temperature, and the reactions initiated bycombining equal volumes of lysate and reaction buffer. Reactions weresampled by quenching individual reaction tubes with 9 volumes acidquench solution, then analyzed as described above.

To assay the combined activity of kinases in lysates (i.e. from NMPs toNTPs), lysates individually expressing each kinase were mixed in a 1:1ratio, divided into 10 aliquots, then heat-inactivated as describedabove. Kinase activity was analyzed in a buffer consisting of 50 mMTris-HCl pH 7.0, 16 mM MgSO₄, 2 mM each nucleotide monophosphate (AMP,CMP, UMP, and GMP), and 16 mM ATP. Reaction buffer (2× concentrate)pre-equilibrated at reaction temperature was combined with an equalvolume of lysate to initiate the reaction. Reactions were performed at70° C. and sampled by quenching individual reaction tubes with 9 volumesacid quench solution, then analyzed as described above.

Polyphosphate Kinase Activity Assays

Polyphosphate kinases were assayed at varying temperatures (37° C., 50°C., 60° C., 70° C., and 80° C.) in a buffer consisting of 50 mM Tris-HClpH 7.0, 4 mM MgSO₄, 25 mM (NH₄)₂SO₄, 1 mM ADP, and 1 mM sodiumhexametaphosphate. Reaction buffer (1.2× concentrate) and enzymesolution (0.25 mg/mL) were pre-equilibrated at reaction temperature for1 minute before reactions were initiated. At time t=0, reactions wereinitiated by mixing 80 μL reaction buffer with 20 μL enzyme. Reactionswere monitored by sampling at the indicated time points. Duringsampling, 15 μL of reaction mixture were transferred to 135 μL of 0.2Msulfuric acid on ice. After completion of the reaction, samples werepH-neutralized with 1M KOH as described above, then diluted 1:10 inice-cold water. ATP was quantified in each sample using the ATPBioluminescent Assay Kit (Sigma-Aldrich), following kit instructions.For reactions in lysates, the above protocol was modified as follows:lysates were aliquoted into individual reaction tubes, thenheat-inactivated by incubating at 70° C. for 15 minutes. Reaction buffer(2× concentrate) and heat-inactivated lysates were pre-equilibrated atreaction temperature, and the reactions initiated by combining equalvolumes of lysate and reaction buffer. Reactions were sampled byquenching individual reaction tubes with 9 volumes acid quench solution,then analyzed as described above. Reaction rates in lysates werecalculated by subtracting signal from a control lysate (withoutoverexpressed polyphosphate kinase) under the same reaction conditions.

Generation of Transcription Templates

Duplex DNA template was prepared by PCR amplification of syntheticgBlock DNA (Integrated DNA Technologies). Reactions were purified andconcentrated by isopropanol precipitation.

RNA Polymerase Downselection

Commercially-available RNA polymerases were compared using conditionsrecommended by each manufacturer. Each 50 μL reaction consisted of 10×reaction buffer (supplied by the manufacturer), NTPs, DNA template (0.5μs), and enzyme. For the NEB T7 RNA polymerase, reactions included 0.5mM each NTP, 5 mM DTT, and 100 U enzyme. Reactions with ThermoT7polymerase were identical, except that DTT was omitted. Reactions withMegaScript T7 included 7.5 mM each NTP and 5 μL enzyme mix. Enzymeconcentrations were determined by BCA assay (Thermo Fisher). Reactionswere monitored by sampling at the indicated time points. Duringsampling, 10 μL of reaction mixture were transferred to 90 μL of RNAquench solution (10 mM Tris-HCl pH 8.0, 5 mM EDTA) and stored on ice.RNA samples in quench solution were quantified using the Quant-iT RNABroad Range Assay Kit (Thermo Fisher), following kit instructions.Serial dilutions of purified dsRNA, prepared using the MegaScript Kitand purified following kit instructions, were used to construct standardcurves for quantitation. Reactions were qualitatively analyzed byagarose gel electrophoresis.

RNA Polymerase Evaluation in Lysates

GL14-322 lysates were heat-inactivated and clarified by centrifugationas described previously. Each 20 μL reaction consisted of clarifiedlysate (7 μL), 10× cofactor solution (300 mM MgCl₂, 20 mM spermidine),NTPs (7.5 mM each, prepared from pH-neutralized stock solutions), DNAtemplate (0.6 μg), and enzyme (1 μL). Reactions were incubated for 1hour at 37° C. or 50° C., then quenched by adding 9 volumes RNA quenchsolution. Quenched reactions were further diluted 10-fold in quenchsolution (final dilution: 100-fold). Diluted reactions were thenquantified using the Quant-iT kit (see above). RNA produced by thereaction was calculated by subtracting RNA quantified in a controlreaction (omitting RNA polymerase).

RNA polymerase assays in high-density lysates were performed asdescribed above, with the following modifications. Each 100 μL reactionconsisted of lysate (67.5 μL), 10× cofactor solution (300 mM MgCl₂, 20mM spermidine), NTPs (7.5 mM each, prepared from pH-neutralized stocksolutions), DNA template (3 μg), and enzyme (10 μL). For reactionsperformed in unclarified reaction matrix, GL14-322 lysates (67.5 μL)were aliquoted into individual reaction tubes, then heat-inactivated asdescribed previously. Additional reactants were added toheat-inactivated matrix, then mixed well by vortexing until homogenous.For reactions performed in buffer, the 10× cofactor solution consistedof 300 mM MgCl₂, 20 mM spermidine, and 400 mM DTT. In addition, 50 mMpotassium phosphate pH 7.4 was included in buffer-only reactions. Allreactions were incubated for 2 hours at 50° C. Samples from eachreaction were quenched by adding 9 volumes RNA quench solution, thenclarified by centrifugation for 1 minute at maximum speed (21,000×g).Supernatants from these reactions were further diluted 40-fold in quenchsolution (final dilution: 400-fold), then quantified using the Quant-iTkit (see above).

Example 2

Thermostable PPK2 enzymes were codon-optimized for expression in E.coli, synthesized, and cloned into pETDuet-1 (Novagen). Plasmids werethen transformed into GL16-170. To generate the Control strain, emptypETDuet-1 plasmid was transformed into GL16-170. After overnightpreculture in 5 mL Lysogeny Broth (LB), strains were cultivated in 1 LLB at 37° C. until cell densities reached an OD₆₀₀ of approximately 0.5.Cultures were then briefly chilled on ice, and PPK2 expression wasinduced by adding isopropyl thiogalactopyranoside (IPTG) to a finalconcentration of 0.25 mM. Post-induction, cultures were grown at 20° C.for approximately 16 hours. Cultures were harvested by centrifugation,and the cell pellets stored at −80° C. Lysates were produced by thawingfrozen pellets, resuspending in 2 pellet volumes 150 mM MOPS-NaOH pH 7,and homogenizing using 4 passes through an EmulsiFlex C3 homogenizer(Avestin) at 15,000 psi. Lysates were then clarified by centrifugationat 15,000×g for 1 hour at 4° C. Aliquots of lysates were frozen at −80°C. before use.

Thermostable PPK2 activity was then measured in heat-inactivatedlysates. Thawed lysates expressing PPK2 enzymes were first diluted 1:100into lysates prepared from the Control strain, except for the D.geothermalis PPK2 lysate, which was diluted 1:10. Pre-chilled solutionsof manganese chloride (MnCl₂) and sodium hexametaphosphate (HMP) wereadded to final concentrations of 10 mM and 1 mM, respectively. Lysateswere then heat-inactivated by incubation in a pre-heated 70° C.thermocycler for 15 minutes. Reactions were then initiated by mixingheat-inactivated lysates with an equal volume of 2× Reaction Buffer,consisting of 10 mM MnCl₂, 2 mM adenosine diphosphate (ADP) or adenosinemonophosphate (AMP), and 9 mM HMP. Reactions were incubated at 70° C.,and time points were taken by removing an aliquot of reaction mixtureand diluting with 9 parts Quench Solution (200 mM H₂SO₄) on ice. Theinitial timepoint (t₀) was taken by directly mixing lysate with QuenchSolution, storing the quenched lysate on ice for 15 minutes, then adding2× reaction buffer. At the conclusion of the assay, quenched timepointsolutions were clarified by centrifugation at 3,200×g for 10 minutes.Supernatants from the quenched reactions were then pH neutralized bymixing 3 parts quenched reaction solution with 1 part NeutralizationSolution (1M KOH). Quenched and neutralized samples were then diluted1:10 with water before quantitation using the Adenosine 5′-triphosphate(ATP) Bioluminescent Assay Kit (Sigma-Aldrich cat #: FLAA), followingkit instructions. Initial reaction rates were calculated based on theaccumulation of ATP in PPK2-containing reactions, subtracting ATPconcentrations from the Control lysate.

Five Class III PPK2 enzymes exhibited soluble expression,thermostability, and high reaction rates in lysates at 70° C.Representative expression and activity data is shown in FIGS. 22A-22B.

A summary of expression and kinetic data for each tested enzyme is shownin Table 17. The C. aerophila, Roseiflexus, A. thermophila, and R.castenholzii enzymes rapidly converted AMP and ADP to ATP using HMP as asubstrate. The D. geothermalis enzyme exhibited conversion rates roughly20× lower than other tested PPK2s for both AMP and ADP substrates.

TABLE 17 Summary of expression and rate data for thermostable Class IIIPPK2 enzymes in lysates. Soluble V_(max) (ADP) V_(max) (AMP) SourceOrganism Expression (mM ATP/hr) (mM ATP/hr) C. aerophila DSM 14535 + 600350 Roseiflexus sp. RS-1 ++ 680 470 A. thermophila UNI-1 ++ 530 480 D.geothermalis DSM 11300 +++ 21 17 R. castenholzii DSM 13941 + 530 370

Example 3

Thermostable C. aerophila PPK2 was then used to supply ATP for cell-freeproduction of dsRNA from NMPs, ADP, and HMP. Cell-free dsRNA synthesisreactions were performed with a mixture of six E. coli lysatesindividually overexpressing the kinases detailed in Table 18.

First, lysates detailed in Table 18 were combined in equal volumes onice. Pre-chilled solutions of manganese chloride (MnCl₂), magnesiumsulfate (MgSO₄), and sodium hexametaphosphate (HMP) were added to finalconcentrations of 0-2.5 mM, 30 mM, and 1 mM, respectively. The lysatemixture was then then heat-inactivated by incubation in a pre-heated 70°C. thermocycler for 15 minutes. To initiate the reactions,heat-inactivated lysates were combined with the following components: anequimolar mixture of nucleotide monophosphates (adenosine5′-monophosphate, cytidine 5′-monophosphate, uridine 5′-monophosphate,and guanosine 5′-monophosphate, 2 mM each), 50 mM Tris pH 7.0, 30 mMMgSO₄, 0-2.5 mM MnCl₂, 1 mM adenosine 5′-diphosphate, 2 mM spermidine,1.5 μg plasmid DNA template, and 3 μg thermostable T7 RNA polymerase(S430P, F849I, F880Y) in a total volume of 20 μL. As a control, dsRNAwas synthesized from an equimolar mixture of 2 mM NTPs (with lysates,ADP, and HMP omitted). As an additional control, dsRNA was synthesizedfrom an equimolar mixture of 2 mM NMPs (with PPK2-expressing lysate,ADP, and HMP omitted, but including 8 mM ATP as an energy source). Asnegative controls, duplicate reactions were performed omittingpolymerase. All reactions were incubated at 50° C. for 2 hours, thenterminated by the addition of 9 volumes TE+ buffer (10 mM Tri s-HCl pH8.0, 5 mM EDTA). Samples were mixed with an equal volume of 2×RNALoading Dye (New England Biolabs) and heated to 70° C. for 10 minutes,followed by agarose/TAE gel electrophoresis.

As shown in FIG. 23, the desired dsRNA product was produced in bufferusing NTPs (left lanes), in nucleotide kinase-expressing lysates fromNMPs and ATP (middle lanes), and in nucleotide kinase and polyphosphatekinase-expressing lysates from NMPs and HMP (right lanes). Manganesechloride was not required in any reaction, demonstrating that the C.aerophila enzyme can utilize Mg²⁺ as a cofactor as well as Mn²⁺.Therefore, Mn²⁺ is not required a priori for cell-free reactionscontaining C. aerophila PPK2.

As shown in FIG. 24, the desired dsRNA product was produced in bufferusing NTPs (left lanes), in nucleotide kinase-expressing lysates fromNMPs and ATP (middle lanes), and in nucleotide kinase and polyphosphatekinase-expressing lysates from NMPs and HMP (right lanes). dsRNAproduction from NMPs and HMPs did not require exogenous ADP or T.thermophilus AMP kinase. Therefore, C. aerophila PPK2 can be used aspart of a 5-kinase system to produce dsRNA from NMPs and HMP incell-free reactions.

OTHER EMBODIMENTS

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

REFERENCES

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Sequences E. coli RNase R (SEQ ID NO: 1)MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGLRRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYLSSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFVVPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGTGMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGEDARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVIPMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTKVWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNAERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIISFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDPENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYSMEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFFVRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNMDERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFRGEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE T. elongatus Ppk (PPK1) (SEQ ID NO: 2)MPSAKSPRRKAPEPIDLDNPQYYFNRSLSWLEFNKRVLHEAYDPRTPLLERLKFMAIFSSNLDEFFMVRVAGLKQQVESGILQVGADGMPPAEQLQAVRQYLLPIVTEQHRYFDQELRALLAKESIFLTRFNELTPEQQAYLNDYFQAQVFPVLTPLAVDPAHPFPYISSLSLNLAVLIRDPESGQERLARVKVPNQFPRFVALPQHLHSPQGVHWLGVPLEEIIAHNLSALFPGMEIEAYFAFRITRSADLELETDKADDLLIAIEQEIRKRRFGSVVRLEVQRGIPPLLRQTLMEEMDLEEIDVYELEGLLCLNDLFAFMGLPLPQFKDPEWQPQVPPSFQRVEERESMFDTSSEITTLGTDYWEAVANELFSLIREGDIIVHHPYHSFAATVQRFITLAAHDPQVLAIKITLYRTSGDSPIVSALIKAAENGKQVAVLVELKARFDEENNILWARKLEKVGVHVVYGVPGLKTHTKTVLVVRQEAGQIRRYVHIGTGNYNPKTASLYEDLGLFSCREELGADLSELFNVLTGYARQRDYRKLLVAPVTMRDRTLQLIYREIEHARNGQPARIIAKMNAITDTQVIRALYEASQAGVDIDLIIRGMCCLRPGVPGVSDRIRVISIIGRFLEHSRIFYFGNNGDPEYYIGSADWRSRNLDRRVEAITPIEDPAIQLELKERLEIMLADNRQAWELQPDGTYRQRQPAPGEAERGTHSVLMARTLKDVQGSH P. furiosus Umk (SEQ ID NO: 3)MRIVFDIGGSVLVPENPDIDFIKEIAYQLTKVSEDHEVAVVVGGGKLARKYIEVAEKFNSSETFKDFIGIQITRANAMLLIAALREKAYPVVVEDFWEAWKAVQLKKIPVMGGTHPGHTTDAVAALLAEFLKADLLVVITNVDGVYTADPKKDPTAKKIKKMKPEELLEIVGKGIEKAGSSSVIDPLAAKIIARSGIKTIVIGKEDAKDLFRVIKGDHNGTTIEP T. thermophilus Cmk(SEQ ID NO: 4)MRGIVTIDGPSASGKSSVARRVAAALGVPYLSSGLLYRAAAFLALRAGVDPGDEEGLLALLEGLGVRLLAQAEGNRVLADGEDLTSFLHTPEVDRVVSAVARLPGVRAWVNRRLKEVPPPFVAEGRDMGTAVFPEAAHKFYLTASPEVRAWRRARERPQAYEEVLRDLLRRDERDKAQSAPAPDALVLDTGGMTLDEVVAWVLAHIRR T. maritima Gmk (SEQ ID NO: 5)MKGQLFVICGPSGAGKTSIIKEVLKRLDNVVFSVSCTTRPKRPHEEDGKDYFFITEEEFLKRVERGEFLEWARVHGHLYGTLRSFVESHINEGKDVVLDIDVQGALSVKKKYSNTVFIYVAPPSYADLRERILKRGTEKEADVLVRLENAKWELMFMDEFDYIVVNENLEDAVEMVVSIVRSERAKVTRNQDKIERFKMEVKGWKKL T. thermophilus Adk (SEQ ID NO: 6)MDVGQAVIFLGPPGAGKGTQASRLAQELGFKKLSTGDILRDHVARGTPLGERVRPIMERGDLVPDDLILELIREELAERVIFDGFPRTLAQAEALDRLLSETGTRLLGVVLVEVPEEELVRRILRRAELEGRSDDNEETVRRRLEVYREKTEPLVGYYEARGVLKRVDGLGTPDEVYARI RAALGIA. aeolicus Ndk (SEQ ID NO: 7)MAVERTLIIVKPDAMEKGALGKILDRFIQEGFQIKALKMFRFTPEKAGEFYYVHRERPFFQELVEFMSSGPVVAAVLEGEDAIKRVREIIGPTDSEEARKVAPNSIRAQFGTDKGKNAIHASDSPESAQYEICFIFSGLEIV Meiothermus ruber DM 1279 PPK2 (SEQ ID NO: 8)MGFCSIEFLMGAQMKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLVVRVKNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE Meiothermus silvanus DSM 9946 PPK2(SEQ ID NO: 9)MAKTIGATLNLQDIDPRSTPGFNGDKEKALALLEKLTARLDELQEQLYAEHQHRVLVILQGMDTSGKDGTIRHVFKNVDPLGVRVVAFKAPTPPELERDYLWRVHQHVPANGELVIFNRSHYEDVLVARVHNLVPPAIWSRRYDHINAFEKMLVDEGTTVLKFFLHISKEEQKKRLLERLVEADKHWKFDPQDLVERGYWEDYMEAYQDVLDKTHTQYAPWHVIPADRKWYRNLQVSRLLVEALEGLRMKYPRPKLNIPRLKSELEKM Deinococcus geothermalis DSM 11300 PPK2(SEQ ID NO: 10)MQLDRYRVPPGQRVRLSNWPTDDDGGLSKAEGEALLPDLQQRLANLQERLYAESQQALLIVLQARDAGGKDGTVKHVIGAFNPSGVQVSNFKVPTEEERAHDFLWRIHRQTPRLGMIGVFNRSQYEDVLVTRVHHLIDDQTAQRRLKHICAFESLLTDSGTRIVKFYLHISPEEQKKRLEARLADPSKHWKFNPGDLQERAHWDAYTAVYEDVLTTSTPAAPWYVVPADRKWFRNLLVSQILVQTLEEMNPQFPAPAFNAADLRIV Thermosynechococcus elongatus BP-1 PPK2(SEQ ID NO: 11)MIPQDFLDEINPDRYIVPAGGNFHWKDYDPGDTAGLKSKVEAQELLAAGIKKLAAYQDVLYAQNIYGLLIIFQAMDAAGKDSTIKHVMSGLNPQACRVYSFKAPSAEELDHDFLWRANRALPERGCIGIFNRSYYEEVLVVRVHPDLLNRQQLPPETKTKHIWKERFEDINHYERYLTRNGILILKFFLHISKAEQKKRFLERISRPEKNWKFSIEDVRDRAHWDDYQQAYADVFRHTSTKWAPWHIIPANHKWFARLMVAHFIYQKLASLNLHYPMLSEAHREQLLEAKALLENEPDEDAnaerolinea thermophila UNI-1 PPK2 (SEQ ID NO: 12)MGEAMERYFIKPGEKVRLKDWSPDPPKDFEGDKESTRAAVAELNRKLEVLQERLYAERKHKVLVILQGMDTSGKDGVIRSVFEGVNPQGVKVANFKVPTQEELDHDYLWRVHKVVPGKGEIVIFNRSHYEDVLVVRVHNLVPPEVWKKRYEQINQFERLLHETGTTILKFFLFISREEQKQRLLERLADPAKHWKFNPGDLKERALWEEYEKAYEDVLSRTSTEYAPWILVPADKKWYRDWVISRVLVETLEGLEIQLPPPLADAETYRRQLLEEDAPESR Caldilinea aerophila DSM 14535 PPK2(SEQ ID NO: 13)MDVDRYRVPPGSTIHLSQWPPDDRSLYEGDKKQGKQDLSALNRRLETLQELLYAEGKHKVLIILQGMDTSGKDGVIRHVFNGVNPQGVKVASFKVPTAVELAHDFLWRIHRQTPGSGEIVIFNRSHYEDVLVVRVHGLVPPEVWARRYEHINAFEKLLVDEGTTILKFFLHISKEEQRQRLLERLEMPEKRWKFSVGDLAERKRWDEYMAAYEAVLSKTSTEYAPWYIVPSDRKWYRNLVISHVIINALEGLNMRYPQPEDIAFDTIVIE Chlorobaculum tepidum TLS PPK2 (SEQ ID NO: 14)MKLDLDAFRIQPGKKPNLAKRPTRIDPVYRSKGEYHELLANHVAELSKLQNVLYADNRYAILLIFQAMDAAGKDSAIKHVMSGVNPQGCQVYSFKHPSATELEHDFLWRTNCVLPERGRIGIFNRSYYEEVLVVRVHPEILEMQNIPHNLAHNGKVWDHRYRSIVSHEQHLHCNGTRIVKFYLHLSKEEQRKRFLERIDDPNKNWKFSTADLEERKFWDQYMEAYESCLQETSTKDSPWFAVPADDKKNARLIVSRIVLDTLESLNLKYPEPSPERRKELLDIRKRLENPENGKOceanithermus profundus DSM 14977 PPK2 (SEQ ID NO: 15)MDVSRYRVPPGSGFDPEAWPTREDDDFAGGKKEAKKELARLAVRLGELQARLYAEGRQALLIVLQGMDTAGKDGTIRHVFRAVNPQGVRVTSFKKPTALELAHDYLWRVHRHAPARGEIGIFNRSHYEDVLVVRVHELVPPEVWGRRYDHINAFERLLADEGTRIVKFFLHISKDEQKRRLEARLENPRKHWKFNPADLSERARWGDYAAAYAEALSRTSSDRAPWYAVPADRKWQRNRIVAQVLVDALEAMDPRFPRVDFDPASVRVE Roseiflexus castenholzii DSM 13941 PPK2(SEQ ID NO: 16)MYAQRVVPGMRVRLHDIDPDANGGLNKDEGRARFAELNAELDVMQEELYAAGIHALLLILQGMDTAGKDGAIRNVMLNLNPQGCRVESFKVPTEEELAHDFLWRVHRVVPRKGMVGVFNRSHYEDVLVVRVHSLVPESVWRARYDQINAFERLLADTGTIIVKCFLHISKEEQEQRLLARERDVSKAWKLSAGDWRERAFWDDYMAAYEEALTRCSTDYAPWYIIPANRKWYRDLAISEALVETLRPYRDDWRRALDAMSRARRAELEAFRAEQHAMEGRPQGAGGVSRR Roseiflexus sp. RS-1 PPK2(SEQ ID NO: 17)MHYAHTVIPGTQVRLRDIDPDASGGLTKDEGRERFASFNATLDAMQEELYAAGVHALLLILQGMDTAGKDGAIRNVMHNLNPQGCRVESFKVPTEEELAHDFLWRVHKVVPRKGMVGVFNRSHYEDVLVVRVHSLVPEHVWRARYDQINAFERLLTDTGTIIVKCFLHISKDEQEKRLLAREQDVTKAWKLSAGDWRERERWDEYMAAYEEALTRCSTEYAPWYIIPANRKWYRDLAISEVLVETLRPYRDDWQRALDAMSQARLAELKAFRHQQTAGATRL Truepera radiovictrix DSM 17093 PPK2(SEQ ID NO: 18)MSQGSAKGLGKLDKKVYARELALLQLELVKLQGWIKAQGLKVVVLFEGRDAAGKGSTIIRITQPLNPRVCRVVALGAPTERERTQWYFQRYVHHLPAAGEMVLFDRSWYNRAGVERVMGFCTEAEYREFLHACPTFERLLLDAGIILIKYWFSVSAAEQERRMRRRNENPAKRWKLSPMDLEARARWVAYSKAKDAMFYHTDTKASPWYVVNAEDKRRAHLSCIAHLLSLIPYEDLTPPPLEMPPRDLAGADEGYERPDKAHQTWVPDYVPPTR

1. A cell-free method of biosynthesizing ribonucleic acid (RNA), themethod comprising: (a) incubating a cell lysate mixture that comprises(i) cellular RNA and (ii) an enzyme that depolymerizes RNA, and (iii) athermostable kinase and/or a thermostable RNA polymerase; and producing,a cell lysate mixture that comprises nucleoside monophosphates; (b)heating the cell lysate mixture produced in step (a) to a temperaturethat inactivates or partially inactivates the enzymes that depolymerizeRNA and enzymes that degrade nucleotides and/or nucleic acids withoutcompletely inactivating the thermostable kinase activities and/orthermostable RNA polymerase activities, and producing a cell lysatemixture that comprises nucleoside monophosphates, and the thermostablekinase and/or the RNA polymerase; and (c) incubating the cell lysatemixture produced in (b) in the presence of an energy source and adeoxyribonucleic acid (DNA) template encoding a RNA of interest,producing nucleoside triphosphates, and producing a cell lysate mixturethat comprises the RNA of interest. 2.-3. (canceled)
 4. The method ofclaim 1, wherein the cellular RNA of step (a) is messenger RNA (mRNA),transfer RNA (tRNA), or ribosomal RNA (rRNA).
 5. The method of claim 1,wherein the enzyme that depolymerizes RNA comprises a ribonuclease. 6.The method of claim 5, wherein the at least one ribonuclease is selectedfrom the group consisting of S1 nuclease, Nuclease P1, RNase II, RNaseIII, RNase R, RNase JI, NucA, PNPase, RNase T, RNase E, and RNaseG. 7.(canceled)
 8. The method of claim 1, wherein the thermostable kinase isselected from the group consisting of thermostable nucleosidemonophosphate kinases, thermostable nucleoside diphosphate kinases, andthermostable polyphosphate kinases.
 9. The method of claim 8, whereinthe thermostable nucleoside monophosphate kinases are selected from thegroup consisting of thermostable uridylate kinases, thermostablecytidylate kinases, thermostable guanylate kinases, and thermostableadenylate kinases.
 10. (canceled)
 11. The method of claim 8, wherein thethermostable nucleoside diphosphate kinases are selected from the groupconsisting of thermostable nucleoside diphosphate kinases encoded by aAquifex aeolicus ndk gene.
 12. The method of claim 1, wherein thethermostable polyphosphate kinases are selected from the groupconsisting of thermostable polyphosphate kinase 1 (PPK1) enzymes andthermostable polyphosphate kinase 2 (PPK2) enzymes. 13.-18. (canceled)19. The method of claim 1, wherein the at least one thermostable RNApolymerase is selected from the group consisting of thermostableDNA-dependent RNA polymerases.
 20. The method of claim 19, wherein thethermostable DNA-dependent RNA polymerases are selected from the groupconsisting of thermostable T7 RNA polymerases, thermostable SP6 RNApolymerases, and thermostable T3 RNA polymerases.
 21. (canceled)
 22. Themethod of claim 1, wherein the energy source is adenosine triphosphate(ATP) or an ATP regeneration system. 23.-27. (canceled)
 28. The methodof claim 1, wherein the cell lysate mixture of step (a) comprises theDNA template encoding the RNA of interest. 29.-38. (canceled)
 39. Themethod of claim 1, wherein the temperature of step (b) is 50° C.-80° C.40.-43. (canceled)
 44. The method of claim 1, wherein the cells arebacterial cells or yeast cells. 45.-50. (canceled)
 51. An engineeredcell comprising an enzyme that depolymerizes RNA, at least onethermostable kinase, and a thermostable RNA polymerase.
 52. The cell ofclaim 51 further comprising an engineered DNA template containing apromoter operably linked to a nucleotide sequence encoding a RNA ofinterest.
 53. A cell lysate produced by lysing the cultured engineeredcell of claim
 51. 54. The cell lysate of claim 53 further comprising anenergy source and nucleoside monophosphates. 55.-88. (canceled)
 89. Themethod of claim 1, the method comprising: (a) incubating a cell lysatemixture that comprises cellular RNA, an enzyme-that depolymerizes RNA,and a thermostable kinase and producing a cell lysate mixture thatcomprises nucleoside monophosphates; (b) heating the cell lysate mixtureproduced in step (a) to a temperature that inactivates or partiallyinactivates endogenous nucleases and phosphatases without completelyinactivating the thermostable kinase, and producing a cell lysatemixture that comprises nucleoside monophosphates, and the thermostablekinase; and (c) incubating the cell lysate mixture produced in (b) inthe presence of an RNA polymerase, an energy source and adeoxyribonucleic acid (DNA) template encoding a RNA of interest,producing nucleoside triphosphates, and producing a cell lysate mixturethat comprises the RNA of interest.
 90. The method of claim 1, themethod comprising: (a) incubating a cell lysate mixture that comprisescellular RNA, an enzyme-that depolymerizes RNA, and a thermostable RNApolymerase and producing a cell lysate mixture that comprises nucleosidemonophosphates; (b) heating the cell lysate mixture produced in step (a)to a temperature that inactivates or partially inactivates endogenousnucleases and phosphatases without completely inactivating thethermostable RNA polymerase, and producing a cell lysate mixture thatcomprises nucleoside monophosphates, and the thermostable RNApolymerase; and (c) incubating the cell lysate mixture produced in (b)in the presence of a kinase, an energy source and a deoxyribonucleicacid (DNA) template encoding a RNA of interest, producing nucleosidetriphosphates, and producing a cell lysate mixture that comprises theRNA of interest.